Mammalian CX3C chemokine genes

ABSTRACT

Nucleic acids encoding a new family of chemokines, the CX3C family, from a mammal, reagents related thereto, including specific antibodies, and purified proteins are described. Methods of using said reagents and related diagnostic kits are also provided.

[0001] The present invention is a continuation of commonly assigned,copending U.S. Ser. No. 08/849,006, filed May 16, 1996, which is acontinuation of commonly assigned then copending U.S. Ser. No.08/590,828 filed Jan. 24, 1996, both of which are incorporated herein byreference.

FIELD OF THE INVENTION

[0002] The present invention contemplates compositions related toproteins which function in controlling development, differentiation,trafficking, and physiology of mammalian cells, e.g., cells of amammalian immune system. In particular, it provides proteins whichregulate or evidence development, differentiation, and function ofvarious cell types, including hematopoietic cells.

BACKGROUND OF THE INVENTION

[0003] The circulating component of the mammalian circulatory systemcomprises various cell types, including red and white blood cells of theerythroid and myeloid cell lineages. See, e.g., Rapaport (1987)Introduction to Hematology (2d ed.) Lippincott, Philadelphia, Pa.; Jandl(1987) Blood: Textbook of Hematology, Little, Brown and Co., Boston,Mass.; and Paul (ed.) (1993) Fundamental Immunology (3d ed.) RavenPress, N.Y.

[0004] For some time, it has been known that the mammalian immuneresponse is based on a series of complex cellular interactions, calledthe “immune network.” Recent research has provided new insights into theinner workings of this network. While it remains clear that much of theresponse does, in fact, revolve around the network-like interactions oflymphocytes, macrophages, granulocytes, and other cells, immunologistsnow generally hold the opinion that soluble proteins, known aslymphokines, cytokines, or monokines, play a critical role incontrolling these cellular interactions. Thus, there is considerableinterest in the isolation, characterization, and mechanisms of action ofcell modulatory factors, an understanding of which should lead tosignificant advancements in the diagnosis and therapy of numerousmedical abnormalities, e.g., immune system and other disorders.

[0005] Lymphokines apparently mediate cellular activities in a varietyof ways. They have been shown to support the proliferation, growth, anddifferentiation of pluripotential hematopoietic stem cells into vastnumbers of progenitors comprising diverse cellular lineages making up acomplex immune system. These interactions between cellular componentsare necessary for a healthy immune response. These different cellularlineages often respond in a different manner when lymphokines areadministered in conjunction with other agents.

[0006] The chemokines are a large and diverse superfamily of proteins.The superfamily is subdivided into three branches, based upon whetherthe first two cysteines in the classical chemokine motif are adjacent(termed the “C-C” branch) or spaced by an intervening residue (“C-X-C”),or a new branch which lacks two cysteines in the corresponding motif,represented by the chemokines known as lymphotactins. See, e.g., Schalland Bacon (1994) Current Opinion in Immunology 6:865-873; and Bacon andSchall (1996) Int. Arch. Allergy & Immunol. 109:97-109.

[0007] Many factors have been identified which influence thedifferentiation process of precursor cells, or regulate the physiologyor migration properties of specific cell types. These observationsindicate that other factors exist whose functions in immune functionwere heretofore unrecognized. These factors provide for biologicalactivities whose spectra of effects may be distinct from knowndifferentiation or activation factors. The absence of knowledge aboutthe structural, biological, and physiological properties of theregulatory factors which regulate cell physiology in vivo prevents themodification of the effects of such factors. Thus, medical conditionswhere regulation of the development or physiology of relevant cells isinappropriate remain unmanageable.

SUMMARY OF THE INVENTION

[0008] The present invention reveals the existence of a previouslyunknown class of chemokine-motif containing molecules which are herebydesignated the CX3C chemokines. The CX3Ckines have three amino acidswhich separate the cysteines in the corresponding region of thechemokine motif. Based on sequence analysis of the two CX3C proteinsequences described below, it is apparent that the CX3Ckines do notbelong to the C, C-C, or C-X-C chemokine families. They represent thefirst known members of a new heretofore unidentified class of chemokinesdesignated CX3Ckines, or alternatively, the CX3C family of chemokines.

[0009] The present invention provides a composition of matter selectedfrom an antibody binding site which specifically binds to a mammalianCX3C chemokine; an expression vector encoding a mammalian CX3C chemokineor fragment thereof; a substantially pure protein which is specificallyrecognized by the antibody binding site; and a substantially pure CX3Cchemokine or peptide thereof, or a fusion protein comprising a 30 aminoacid fragment of CX3C chemokine sequence.

[0010] In the antibody binding site embodiments, the antibody bindingsite may be: specifically immunoreactive with a mature protein selectedfrom the group consisting of the polypeptides of SEQ ID NO: 2, 4, 6 and8; raised against a purified or recombinantly produced human or mouseCX3C chemokine; in a monoclonal antibody, Fab, or F(ab)2; immunoreactivewith denatured antigen; or in a labeled antibody. In certainembodiments; the antibody binding site is detected in a biologicalsample by a method of: contacting a binding agent having an affinity forCX3C chemokine protein with the biological sample; incubating thebinding agent with the biological sample to form a binding agent:Cx3Cchemokine protein complex; and detecting the complex. In a preferredembodiment, the biological sample is human, and the binding agent is anantibody.

[0011] A kit embodiment is provided possessing a composition, describedabove, with either instructional material for the use of thecomposition; or segregation of the composition into a container.

[0012] A nucleic acid embodiment of the invention includes an expressionvector encoding a CX3C chemokine protein, wherein the proteinspecifically binds an antibody generated against an immunogen selectedfrom the mature polypeptide portions of SEQ ID NO: 2, 4, 6, and 8. Thevector may: encode a CX3C chemokine polypeptide with complete sequenceidentity to a naturally occurring human CX3C chemokine domain; encode aCX3C chemokine protein comprising sequence selected from thepolypeptides of SEQ ID NO: 2, 4, 6, and 8; or comprise sequence selectedfrom the nucleic acids of SEQ ID NO: 1, 3, 5, or 7. In otherembodiments, the vector is capable of selectively hybridizing to anucleic acid encoding a CX3C chemokine protein, e.g., a mature proteincoding segment of SEQ ID NO: 1, 3, 5, or 7. In various preferredembodiments, the isolated nucleic acid is detected in a biologicalsample by a method: contacting a biological sample with a nucleic acidprobe capable of selectively hybridizing to the nucleic acid; incubatingthe nucleic acid probe with the biological sample to form a hybrid ofthe nucleic acid probe with complementary nucleic acid sequences presentin the biological sample; and determining the extent of hybridization ofthe nucleic acid probe to the complementary nucleic acid sequences. Insuch method, preferably the nucleic acid probe is capable of hybridizingto a nucleic acid encoding a protein consisting of the polypeptides ofSEQ ID NO: 2, 4, 6, or 8.

[0013] In protein embodiments, the isolated CX3C chemokine protein willpreferably be of approximately 11,000 to 15,000 daltons when inunglycosylated form, and the CX3C chemokine protein specifically bindsto an antibody generated against an immunogen; the polypeptides of SEQID NO: 2, 4, 6, or 8; and the CX3C chemokine lacks the cysteinestructural motifs and sequence characteristic of a C, a CC, or a CXCchemokine. In various embodiments, the isolated CX3C chemokine proteinis: selected from human CX3Ckine or mouse CX3Ckine; consists of apolypeptide comprising sequence from SEQ ID NO: 2, 4, 6, or 8;recombinantly produced, or a naturally occurring protein.

[0014] The present invention also embraces a cell transfected with thenucleic acid encoding a CX3C chemokine, e.g., where the nucleic acid hasSEQ ID NO: 1, 3, 5, or 7.

[0015] The invention also provides a method of modulating physiology ordevelopment of a cell by contacting the cell with a CX3C chemokine, oran antagonist of the chemokine. In preferred embodiments, the physiologyis attraction, and the cell is a peripheral blood monocyte or a T cell.

DETAILED DESCRIPTION

[0016] I. General

[0017] The present invention provides DNA sequences encoding mammalianproteins which exhibit structural properties or motifs characteristic ofa cytokine or chemokine. For a review of the chemokine family, see,e.g., Lodi, et al. (1994) Science 263:1762-1767; Gronenborn and Clore(1991) Protein Engineering 4:263-269; Miller and Kranger (1992) Proc.Nat'l Acad. Sci. USA 89:2950-2954; Matsushima and Oppenheim (1989)Cytokine 1:2-13; Stoeckle and Baker (1990) New Biol. 2:313-323;Oppenheim, et al. (1991) Ann. Rev. Immunol. 9:617-648; Schall (1991)Cytokine 3:165-183; and The Cytokine Handbook Academic Press, NY. Theproteins described herein are designated CX3Ckines because they wereinitially recognized as sharing significant structural features ofchemokines, but whose structural features also exhibit sequencepeculiarity, e.g., structural motifs, distinct from the other knownbranches of the chemokine molecules.

[0018] The best characterized embodiment of this family of proteins wasdiscovered from a human and is designated human CX3C chemokine (GenBankAccession No. H14940). See, SEQ ID NO: 1-4 An additional CX3Ckine,represented by a mouse molecule, designated mouse CX3Ckine, is-alsodescribed herein. See Table 1 and SEQ ID NO: 5-8. The descriptions beloware directed, for exemplary purposes, to primate and rodent embodiments,e.g., human and mouse, but are likewise applicable to relatedembodiments from other, e.g., natural, sources. These sources shouldinclude various vertebrates, typically warm blooded animals, e.g., birdsand mammals, particularly domestic animals, and primates.

[0019] In the human sequence (SEQ ID NO: 1-4), the signal sequence runsfrom about Met1 to Gly24, thus the mature polypeptide begins at aboutGln25 and ends at about Val 397. A chemokine domain runs from aboutGln25 to about GlylOO; a stalk region, which possesses many potentialglycosylation sites, runs from about Gly101 to about Gln341; atranmembrane region begins at about Ala342 and ends at about Thr361; andan intracellular domain, containing two tyrosine phosphorylation sitesat residues 382 and 392, runs from about Tyr362 to Val397. TABLE 1 MouseCX3C chemokine nucleic acid (SEQ ID NO: 7) and amino acid (SEQ ID NO: 8)sequences. The coding sequence runs from nucleotides 62-1249. The signalsequence runs from about Met1 through Gly24. The mature polypeptide runsfrom about Gln25 through Val395. The chemokine domain runs from aboutGln25 through Gly100. The stalk region runs from about Gly101 throughGln339. The transmembrane domain runs from about Ala340 through Phe358.The cytoplasmic domain runs from about Ala359 through Val395. TGACTACTAGGAGCTGCGAC ACGGCCCAGC CTCCTGGCCG CCGAATTCCT GCACTCCAGC 60 C ATG GCT CCCTCG CCG CTC GCG TGG CTG CTG CGC CTG GCC GCG TTC 106   Met Ala Pro SerPro Leu Ala Trp Leu Leu Arg Leu Ala Ala Phe    1               5                  10                  15 TTC CATTTG TGT ACT CTG CTG CCG GGT CAG CAC CTC GGC ATG ACG AAA 154 Phe His LeuCys Thr Leu Leu Pro Gly Gln His Leu Gly Met Thr Lys                 20                  25                  30 TGC GAA ATCATG TGC GGC AAG ATG ACC TCA CGA ATC CCA GTG GCT TTG 202 Cys Glu Ile MetCys Gly Lys Met Thr Ser Arg Ile Pro Val Ala Leu             35                  40                  45 CTC ATC CGC TATCAG CTA AAT CAG GAG TCC TGC GGC AAG CGT GCC ATT 250 Leu Ile Arg Tyr GlnLeu Asn Gln Glu Ser Cys Gly Lys Arg Ala Ile         50                  55                  60 GTC CTG GAG ACG ACACAG CAC AGA CGC TTC TGT GCT GAC CCG AAG GAG 298 Val Leu Glu Thr Thr GlnHis Arg Arg Phe Cys Ala Asp Pro Lys Glu     65                  70                  75 AAA TGG GTC CAA GAC GCCATG AAG CAT CTG GAT CAC CAG GCT GCT GCC 346 Lys Trp Val Gln Asp Ala MetLys His Leu Asp His Gln Ala Ala Ala 80                  85                  90                  95 CTC ACTAAA AAT GGT GGC AAG TTT GAG AAG CGG GTG GAC AAT GTG ACA 394 Leu Thr LysAsn Gly Gly Lys Phe Glu Lys Arg Val Asp Asn Val Thr                100                 105                 110 CCT GGG ATCACC TTG GCC ACT AGG GGA CTG TCC CCA TCT GCC CTG ACA 442 Pro Gly Ile ThrLeu Ala Thr Arg Gly Leu Ser Pro Ser Ala Leu Thr            115                 120                 125 AAG CCT GAA TCCGCC ACA TTG GAA GAC CTT GCT TTG GAA CTG ACT ACT 490 Lys Pro Glu Ser AlaThr Leu Glu Asp Leu Ala Leu Glu Leu Thr Thr        130                 135                 140 ATT TCC CAG GAG GCCAGG GGG ACC ATG GGG ACT TCC CAA GAG CCA CCG 538 Ile Ser Gln Glu Ala ArgGly Thr Met Gly Thr Ser Gln Glu Pro Pro    145                 150                 155 GCA GCA GTG ACC GGA TCATCT CTC TCA ACT TCC GAG GCA CAG GAT GCA 586 Ala Ala Val Thr Gly Ser SerLeu Ser Thr Ser Glu Ala Gln Asp Ala160                 165                 170                 175 GGG CTTACG GCT AAG CCT CAG AGC ATT GGA AGT TTT GAG GCG GCT GAC 634 Gly Leu ThrAla Lys Pro Gln Ser Ile Gly Ser Phe Glu Ala Ala Asp                180                 185                 190 ATC TCC ACCACC GTT TGG CCG AGT CCT GCT GTC TAC CAA TCT GGA TCT 682 Ile Ser Thr ThrVal Trp Pro Ser Pro Ala Val Tyr Gln Ser Gly Ser            195                 200                 205 AGC TCC TGG GCTGAG GAA AAA GCT ACT GAG TCC CCC TCC ACT ACA GCC 730 Ser Ser Trp Ala GluGlu Lys Ala Thr Glu Ser Pro Ser Thr Thr Ala        210                 215                 220 CCA TCT CCT CAG GTGTCC ACT ACT TCA CCT TCA ACC CCA GAG GAA AAT 778 Pro Ser Pro Gln Val SerThr Thr Ser Pro Ser Thr Pro Glu Glu Asn    225                 230                 235 GTT GGG TCC GAA GGC CAACCC CCA TGG GTC CAG GGA CAG GAG CTC AGT 826 Val Gly Ser Glu Gly Gln ProPro Trp Val Gln Gly Gln Asp Leu Ser240                 245                 250                 255 CCA GAGAAG TCT CTA GGG TCT GAG GAG ATA AAC CCA GTT CAT ACT GAT 874 Pro Glu LysSer Leu Gly Ser Glu Glu Ile Asn Pro Val His Thr Asp                260                 265                 270 AAT TTC CAGGAG AGG GGG CCT GGC AAC ACA GTC CAC CCC TCA GTG GCT 922 Asn Phe Gln GluArg Gly Pro Gly Asn Thr Val His Pro Ser Val Ala            275                 280                 285 CCC ATC TCC TCTGAA GAG ACC CCC AGC CCA GAG CTG GTG GCC TCG GGC 970 Pro Ile Ser Ser GluGlu Thr Pro Ser Pro Glu Leu Val Ala Ser Gly        290                 295                 300 AGC CAG GCT CCT AAGATA GAG GAA CCC ATC CAT GCC ACT GCA GAT CCC 1018 Ser Gln Ala Pro Lys IleGlu Glu Pro Ile His Ala Thr Ala Asp Pro    305                 310                 315 CAG AAA CTG AGT GTG CTTATC ACT CCT GTC CCC GAC ACC CAG GCA GCC 1066 Gln Lys Leu Ser Val Leu IleThr Pro Val Pro Asp Thr Gln Ala Ala320                 325                 330                 335 ACA AGGAGG CAG GCA GTG GGG CTA CTG GCT TTC CTT GGT CTT CTT TTC 1114 Thr Arg ArgGln Ala Val Gly Leu Leu Ala Phe Leu Gly Leu Leu Phe                340                 345                 350 TGC CTA GGGGTG GCC ATG TTT GCT TAC CAG AGC CTT CAG GGC TGT CCC 1162 Cys Leu Gly ValAla Met Phe Ala Tyr Gln Ser Leu Gln Gly Cys Pro            355                 360                 365 CGC AAA ATG GCGGGG GAA ATG GTA GAA GGC CTC CGC TAC GTC CCC CGT 1210 Arg Lys Met Ala GlyGlu Met Val Glu Gly Leu Arg Tyr Val Pro Arg        370                 375                 380 AGC TGT GGC AGT AACTCA TAC GTC CTG GTG CCA GTG TGA GCTGCTTGCC 1259 Ser Cys Gly Ser Asn SerTyr Val Leu Val Pro Val  *    385                 390                 395 TGCCTGCCTG TGTCCAGAGTGTGATTCGGA CAGCTGTCTG GGGACCCCCC CCCATCCTCA 1319 TACCCACCTT CATCCACGCTGGGGAAATGG GAATGGAGAA GCTGGACCTC CAGGGGCTGT 1379 GGGCTCCATC CAATCCCCCTTCCCCCGAGG GGTGGCCCCG GAGGCCACCC TAGACCACTA 1439 TTCACTTATC AGAGACAGAGCAGGTGACCT TCCAGCTCCT CTATATTTGA AAGAATCCTC 1499 TGCTGCTGGC TGGTTAGAGGGGCCCTTGAC ACCCCAACTC CAGTGAACAA TTATTTATTG 1559 GATTCCCAGC CCCTGCGACGACACCTGTTT CCCGCGCGCA CCGTGGTCCG CCCATATCAC 1619 AAGCAGCAGG CCAGGCCTATCTGCCTGTCC CCCTGACCTC CTTGTGTCTC CTGGCTTTGC 1679 TGCAGTCGCC AGCCCTTCTCCTCCCCGGCC AGCCGCGGTG CTATCTGCCC TATGTCTCCC 1739 TCTATCCCCT GTACAGAGCGCACCACCATC ACCATCAACA CCGCTGTTGT GTCTTTTCTT 1799 GCATGAGGTT AAAGCTGTGTTTTCTGGAGC TCTCCGGGAA GGGAGACAAG CTTGCGAGAG 1859 GGTTTAAAGT GTTCCTCCCCAGACTTGGAT GTGCTGTGAG GGCATGCTGC GTCTGAAGGA 1919 AGGGTCCAGT CCCCACTCGGCTACCAGCAC CACAAAGTGC CCCACCTGTA AAAGGAAAGA 1979 AACGTGGTCC AGAGCTGGCAATAACCTATG GCCCTGACAT CATCACTTTC TCTGAGATCC 2039 TTGTCTCCAC CCCTGGGTGCAACCCCACCC CTTATCAACA TTAATAGTCA CTGCCATTCC 2099 ACTGGACTGA CATTTTTGTACCCTGTGATT CTGAGGGCTG GCAAGGAGTG GCTTGAGAGT 2159 GCAGATCGTA CCCTGTATGCCCCCCCCAAA TGGAGGCTGA GTTGGGGACT TGCAGGAACA 2219 GAGGCCAACT CAGATGGCTTCCCCTGTGTT CTCACTAGAA ACCCCTCCCC CATGCACCAA 2279 GGTGACAGTC ACAGGTCTGCCCTGGCTAAA GGACAAGCCA CATAGGAAAG ATTAGGACAA 2339 GCCCCTCGGA GGCAGAGGATCCAGGGTAAA CCCCTGGAGT GGCCACAAAC CCAATTTCAG 2399 TGTAGGGACT TGTGCATGTGTGTACTTGCA TAGTCAGACA GAGGCTGCCA GGGTCCTTTC 2459 CTGTCTCTGA GAGCAGTGTTCACGCCAAGG ACTCACCTTT GCCCCCATTG CAGGCAGGGC 2519 CAGAACTCCC ATAGCATTCTCCAAGAGCCC TGTGACATTT TCTGGAAGGA ACTCTGCCCT 2579 GGGCGCAAAG TGACTGCTGAAGCAAGGAGC AGCTGAGCAG CACCCCAGCG GAGCTGAGCC 2639 GGCAGGCCAC GCCCCTCGGGGGGGGGCATT TCTACCCGCC CTGCTCTGAA TAGCTCCAAC 2699 TTCACCTTAG GAGCCTCCCAGGGGCGAGCT TCACCCAGAA GCCAGTGACT CACTCCTTGA 2759 TTGGTGGAAG CTCAGTTGGCTCCTGAGAGT GAGGAAGCCA ACCCTTTGTC GACCCTCCTC 2819 CTGGGAAGCC TGTGGGCGGCTCTGATCATG CTCCACAGAA CCAGTTGTAG GCCTGAGCCG 2879 CAGCAGCCCG AGTGCACTATATCCTGGCTC CTTCGGTGGG GAACCTTTAA GGGTTGGGAC 2939 ACCCGTCATC GGACTTTGTTGGTTCCTCCC TCCCAGAGCA GAATGTGGGC CGTAACAATC 2999 TGAGGAGGAC TTTAAAAGTTGTTGATCCTT TAGGGTTTTT TTTCAAGCAT CATTACCAAT 3059 GTCTGT     3065

[0020] The CX3Ckine proteins of this invention are defined in part bytheir physicochemical and biological properties. The biologicalproperties of the human and mouse CX3Ckines described herein, e.g.,human CX3Ckine and mouse CX3Ckine, are defined by their amino acidsequence, and mature size. They also should share biological properties.The human and mouse CX3Ckine molecules exhibit about 70-80% amino acididentity, depending on whether the signal or mature sequences arecompared. One of skill will readily recognize that some sequencevariations may be tolerated, e.g., conservative substitutions orpositions remote from the helical structures, without alteringsignificantly the biological activity of the molecule.

[0021] Table 2 shows a sequence alignment of human CX3Ckine amino acidsequence (CX3C) with the C-X-C chemokine Groα (Gro), the C chemokinelymphotactin (LTn), and the C-C chemokine Macrophage inflammatoryprotein 1β (MIP-1β). TABLE 2 Comparison of various chemokines Exon 1 GroMIPATRSLLCAALLLLATSRLATG                    (SEQ ID NO: 9) LTnMRLLLLTFLGVCCLTPWVV                         (SEQ ID NO: 10) MIP-1βMKLCVSALSLLLLVAAFCAPGFS                     (SEQ ID NO: 11) CX3MAPISLSWLLRLATFCHLTVLLAG                    (SEQ ID NO: 2) Exon 2 GroAPIANELRCQCLQTMA.GIHLKNIQSLKVLPSGPHCTQT LTnEGVGTEVLEESSCVNLQTQRLPVQKIKTYIIWEG....AMR MIPAPMGSDPPTSCCFSYTARKLPRNFVVDYYETSSL...CSQP CX3QHHGVTKCNITC.SKMTSKIPVALLIHYQQNQAS...CGKR Exon 3 GR0EVIATLKNGREACLDPEAPLVQKIVQKMLKGVPK LTNAVIFVTKRGLKICADPEAKWVLAAIKTVDGRASTRKNMAETVPGTGAQRSTSTAITLTG MIPAVVFQTKRSKQVCADPSESWVQEYVYDLELN CX3AIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTRNG...

[0022] CX3Ckines are present in specific tissue types, e.g., neuraltissues, and the interaction of the protein with a receptor will beimportant for mediating various aspects of cellular physiology ordevelopment. The cellular types which express message encoding CX3Ckinessuggest that signals important in cell differentiation and developmentare mediated by them. See, e.g., Gilbert (1991) Developmental Biology(3d ed.) Sinauer Associates, Sunderland, MA; Browder, et al. (1991)Developmental Biology (3d ed.) Saunders, Philadelphia, Pa.; Russo, etal. (1992) Development: The Molecular Genetic Approach Springer-Verlag,New York, N.Y.; and Wilkins (1993) Genetic Analysis of AnimalDevelopment (2d ed.) Wiley-Liss, New York, N.Y. Moreover, CX3Ckineexpression should serve to define certain cell subpopulations.

[0023] The CX3C chemokine producing profile of various cells iselucidated herein. Screening a cDNA library generated from brainprovided a novel cytokine, designated human CX3Ckine. Human CX3Ckineexhibits distant similarity to members of the C, C-C, and C-X-Cchemokine families, with another heretofore unrecognized number of aminoacid residues separating the characteristic cysteines in the motif whichis peculiar to and partially defines chemokines. These observationssuggest that the CX3Ckines represent novel additions to the chemokinesuperfamily.

[0024] CX3C chemokine protein biochemistry was assessed in mammalianexpression systems. Human embryonic kidney 293 cells (HEK 293)transfected with a mammalian expression construct encoding full-lengthCX3C chemokine were metabolically labeled with 35S cysteine andmethionine. CX3C chemokine was produced as a protein of Mr ˜95 kDa;control transfected supernatants contained no such species.Neuraminidase and glycosidases reduced the Mr of CX3C chemokine from ˜95kDa to ˜45 kDa, suggesting that the recombinant form, is glycosylatedsubstantially. CX3C chemokine cDNA, encoding a predicted membrane-boundprotein, encodes a glycoprotein which is released from cells by anundefined mechanism.

[0025] The pro-migratory activities of CX3C chemokine have been assessedin microchemotaxis assays. CX3C chemokine appears to be a potentattractant of peripheral blood monocytes and T cells. Pro-migratoryactivity for blood neutrophils has been difficult to demonstrate.

[0026] The CX3C chemokine gene has been mapped to human chromosome 16.Mapping studies also indicate the possibility of a pseudogene or relatedgene on human chromosome 14. Sequencing of genomic DNA fragmentssuggests CX3C chemokine gene has an intron which begins near or in thecodon encoding Ile 64. Other intron/exon boundaries have yet to bemapped, but such will be easily accomplished by standard methods.

[0027] The membrane bound form of CX3Ckine possesses proadherentproperties for circulating T cells and monocytes. A secreted or solubleform, consisting of the chemokine domain and the stalk region, is ableto inhibit this proadhesive activity. This suggests that the membranebound form of CX3Ckine may be a potent regulator of circulatingleukocytes, and thus may be involved in various inflammatory diseases,e.g., arthritis. The soluble form may be used as a regulator ofproadherence, especially in conditions of compromised immune response.

[0028] CX3C chemokine's properties as a T cell and monocytechemoattractant, coupled with its distribution in brain and otherorgans, suggests that CX3C chemokine may be involved in the pathogenesisof such CNS inflammatory disorders as multiple sclerosis, and otherpathologies involving neurogenic inflammation. Since CX3C chemokinedistribution is not limited to the brain, however, the entire spectrumof inflammatory, infectious, and immunoregulatory states thought toinvolve other chemokines must also now be considered for CX3C chemokine.See, e.g., Frank, Et al. (eds.) (1995) Samter's Immnologic Diseases 5thed., vols. I and II, Little, Brown, and Co., Boston, Mass.

[0029] II. Definitions

[0030] The term “binding composition” refers to molecules that bind withspecificity to a CX3Ckine, e.g., in an antibody-antigen interaction.However, other compounds, e.g., receptor proteins, may also specificallyassociate with CX3Ckines to the exclusion of other molecules. Typically,the association will be in a natural physiologically relevantprotein-protein interaction, either covalent or non-covalent, and mayinclude members of a multiprotein complex, including carrier compoundsor dimerization partners. The molecule may be a polymer, or chemicalreagent. No implication as to whether a CX3Ckine is either the ligand orthe receptor of a ligand-receptor interaction is necessarilyrepresented, other than whether the interaction exhibits similarspecificity, e.g., specific affinity. A functional analog may be aligand with structural modifications, or may be a wholly unrelatedmolecule, e.g., which has a molecular shape which interacts with theappropriate ligand binding determinants. The ligands may serve asagonists or antagonists of the receptor, see, e.g., Goodman, et al.(eds.) (1990) Goodman & Gilman's: The Pharmacological Bases ofTherapeutics (8th ed.) Pergamon Press, Tarrytown, N.Y.

[0031] The term “binding agent:CX3Ckine protein complex”, as usedherein, refers to a complex of a binding agent and a CX3Ckine proteinthat is formed by specific binding of the binding agent to the CX3Ckineprotein, e.g., preferably the chemokine domain. Specific binding of thebinding agent means that the binding agent has a specific binding sitethat recognizes a site on the CX3Ckine protein. For example, antibodiesraised to a CX3Ckine protein and recognizing an epitope on the CX3Ckineprotein are capable of forming a binding agent:CX3Ckine protein complexby specific binding. Typically, the formation of a bindingagent:CX3Ckine protein complex allows the measurement of CX3Ckineprotein in a mixture of other proteins and biologics. The term“antibody:CX3Ckine protein complex” refers to an embodiment in which thebinding agent is an antibody. The antibody may be monoclonal,polyclonal, or a binding fragment of an antibody, e.g, an Fab of F(ab)2fragment. The antibody will preferably be a polyclonal antibody forcross-reactivity purposes.

[0032] “Homologous” nucleic acid sequences, when compared, exhibitsignificant similarity. The standards for homology in nucleic acids areeither measures for homology generally used in the art by sequencecomparison and/or phylogenetic relationship, or based upon hybridizationconditions. Hybridization conditions are described in greater detailbelow.

[0033] An “isolated” nucleic acid is a nucleic acid, e.g., an RNA, DNA,or a mixed polymer, which is substantially separated from other biologiccomponents which naturally accompany a native sequence, e.g., proteinsand flanking genomic sequences from the originating species. The termembraces a nucleic acid sequence which has been removed from itsnaturally occurring environment, and includes recombinant or cloned DNAisolates and chemically synthesized analogs, or analogs biologicallysynthesized by heterologous systems. A substantially pure moleculeincludes isolated forms of the molecule. An isolated nucleic acid willusually contain homogeneous nucleic acid molecules, but will, in someembodiments, contain nucleic acids with minor sequence heterogeneity.This heterogeneity is typically found at the polymer ends or portionsnot critical to a desired biological function or activity.

[0034] As used herein, the term “CX3Ckine protein” shall encompass, whenused in a protein context, a protein having amino acid sequences,particularly from the chemokine motif portions, shown in SEQ ID NO: 2,4, 6, or 8, or a significant fragment of such a protein, e.g.,preferabley the chemokine domain. The invention also embraces apolypeptide which exhibits similar structure to human or mouse CX3Ckine,e.g., which interacts with CX3Ckine specific binding components. Thesebinding components, e.g., antibodies, typically bind to a CX3Ckine withhigh affinity, e.g., at least about 100 nM, usually better than about 30nM, preferably better than about 10 nM, and more preferably at betterthan about 3 nM.

[0035] The term “polypeptide” or “protein” as used herein includes asignificant fragment or segment of chemokine motif portion of aCX3Ckine, and encompasses a stretch of amino acid residues of at leastabout 8 amino acids, generally at least 10 amino acids, more generallyat least 12 amino acids, often at least 14 amino acids, more often atleast 16 amino acids, typically at least 18 amino acids, more typicallyat least 20 amino acids, usually at least 22 amino acids, more usuallyat least 24 amino acids, preferably at least 26 amino acids, morepreferably at least 28 amino acids, and, in particularly preferredembodiments, at least about 30 or more amino acids, e.g., 35, 40, 45,50, 60, 70, 80, etc.

[0036] A “recombinant” nucleic acid is defined either by its method ofproduction or its structure. In reference to its method of production,e.g., a product made by a process, the process is use of recombinantnucleic acid techniques, e.g., involving human intervention in thenucleotide sequence, typically selection or production.

[0037] Alternatively, it can be a nucleic acid made by generating asequence comprising fusion of two fragments which are not naturallycontiguous to each other, but is meant to exclude products of nature,e.g., naturally occurring mutants. Thus, for example, products made bytransforming cells with any non-naturally occurring vector isencompassed, as are nucleic acids comprising sequence derived using anysynthetic oligonucleotide process. Such is often done to replace a codonwith a redundant codon encoding the same or a conservative amino acid,while typically introducing or removing a sequence recognition site.Alternatively, it is performed to join together nucleic acid segments ofdesired functions to generate a single genetic entity comprising adesired combination of functions not found in the commonly availablenatural forms. Restriction enzyme recognition sites are often the targetof such artificial manipulations, but other site specific targets, e.g.,promoters, DNA replication sites, regulation sequences, controlsequences, or other useful features may be incorporated by design. Asimilar concept is intended for a recombinant, e.g., fusion,polypeptide. Specifically included are synthetic nucleic acids which, bygenetic code redundancy, encode polypeptides similar to fragments ofthese antigens, and fusions of sequences from various different speciesvariants. Mutation of protease cleavage sites may also be accomplished.

[0038] “Solubility” is reflected by sedimentation measured in Svedbergunits, which are a measure of the sedimentation velocity of a moleculeunder particular conditions. The determination of the sedimentationvelocity was classically performed in an analytical ultracentrifuge, butis typically now performed in a standard ultracentrifuge. See,Freifelder (1982) Physical Biochemistry (2d ed.) W.H. Freeman & Co., SanFrancisco, Calif.; and Cantor and Schimmel (1980) Biophysical Chemistryparts 1-3, W.H. Freeman & Co., San Francisco, Calif. As a crudedetermination, a sample containing a putatively soluble polypeptide isspun in a standard full sized ultracentrifuge at about 50K rpm for about10 minutes, and soluble molecules will remain in the supernatant. Asoluble particle or polypeptide will typically be less than about 30S,more typically less than about 15S, usually less than about 10S, moreusually less than about 6S, and, in particular embodiments, preferablyless than about 4S, and more preferably less than about 3S. Solubilityof a polypeptide or fragment depends upon the environment and thepolypeptide. Many parameters affect polypeptide solubility, includingtemperature, electrolyte environment, size and molecular characteristicsof the polypeptide, and nature of the solvent. Typically, thetemperature at which the polypeptide is used ranges from about 4° C. toabout 65° C. Usually the temperature at use is greater than about 18° C.and more usually greater than about 22° C. For diagnostic purposes, thetemperature will usually be about room temperature or warmer, but lessthan the denaturation temperature of components in the assay. Fortherapeutic purposes, the temperature will usually be body temperature,typically about 37° C. for humans, though under certain situations thetemperature may be raised or lowered in situ or in vitro.

[0039] The size and structure of the polypeptide should generally beevaluated in a substantially stable state, and usually not in adenatured state. The polypeptide may be associated with otherpolypeptides in a quaternary structure, e.g., to confer solubility, orassociated with lipids or detergents in a manner which approximatesnatural lipid bilayer interactions.

[0040] The solvent will usually be a biologically compatible buffer, ofa type used for preservation of biological activities, and will usuallyapproximate a physiological solvent. Usually the solvent will have aneutral pH, typically between about 5 and 10, and preferably about 7.5.On some occasions, a detergent will be added, typically a mildnon-denaturing one, e.g., CHS (cholesteryl hemisuccinate) or CHAPS(3-[3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate), or a lowenough concentration as to avoid significant disruption of structural orphysiological properties of the protein.

[0041] “Substantially pure” in a protein context typically means thatthe protein is isolated from other contaminating proteins, nucleicacids, and other biologicals derived from the original source organism.Purity, or “isolation” may be assayed by standard methods, and willordinarily be at least about 50% pure, more ordinarily at least about60% pure, generally at least about 70% pure, more generally at leastabout 80% pure, often at least about 85% pure, more often at least about90% pure, preferably at least about 95% pure, more preferably at leastabout 98% pure, and in most preferred embodiments, at least 99% pure.Similar concepts apply, e.g., to antibodies or nucleic acids.

[0042] “Substantial similarity” in the nucleic acid sequence comparisoncontext means either that the segments, or their complementary strands,when compared, are identical when optimally aligned, with appropriatenucleotide insertions or deletions, in at least about 50% of thenucleotides, generally at least 56%, more generally at least 59%,ordinarily at least 62%, more ordinarily at least 65%, often at least68%, more often at least 71%, typically at least 74%, more typically atleast 77%, usually at least 80%, more usually at least about 85%,preferably at least about 90%, more preferably at least about 95 to 98%or more, and in particular embodiments, as high at about 99% or more ofthe nucleotides. Alternatively, substantial similarity exists when thesegments will hybridize under selective hybridization conditions, to astrand, or its complement, typically using a sequence derived from SEQID NO: 1, 3, 5 or 7. Typically, selective hybridization will occur whenthere is at least about 55% similarity over a stretch of at least about30 nucleotides, preferably at least about 65% over a stretch of at leastabout 25 nucleotides, more preferably at least about 75%, and mostpreferably at least about 90% over about 20 nucleotides. See Kanehisa(1984) Nuc. Acids Res. 12:203-213. The length of similarity comparison,as described, may be over longer stretches, and in certain embodimentswill be over a stretch of at least about 17 nucleotides, usually atleast about 20 nucleotides, more usually at least about 24 nucleotides,typically at least about 28 nucleotides, more typically at least about40 nucleotides, preferably at least about 50 nucleotides, and morepreferably at least about 75 to 100 or more nucleotides, e.g., 150, 200,etc.

[0043] “Stringent conditions”, in referring to homology or substantialsimilarity in the hybridization context, will be stringent combinedconditions of salt, temperature, organic solvents, and other parameters,typically those controlled in hybridization reactions. The combinationof parameters is more important than the measure of any singleparameter. Stringent temperature conditions will usually includetemperatures in excess of about 30° C., more usually in excess of about37° C., typically in excess of about 45° C., more typically in excess ofabout 55° C., preferably in excess of about 65° C., and more preferablyin excess of about 70° C. Stringent salt conditions will ordinarily beless than about 1000 mM, usually less than about 500 mM, more usuallyless than about 400 mM, typically less than about 300 mM, preferablyless than about 200 mM, and more preferably less than about 150 mM. See,e.g., Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370. A nucleicacid probe which binds to a target nucleic acid under stringentconditions is specific for said target nucleic acid. Such a probe istypically more than 11 nucleotides in length, and is sufficientlyidentical or complementary to a target nucleic acid over the regionspecified by the sequence of the probe to bind the target understringent hybridization conditions.

[0044] CX3Ckines from other mammalian species can be cloned and isolatedby cross-species hybridization of closely related species. See, e.g.,below. Similarity may be relatively low between distantly relatedspecies, and thus hybridization of relatively closely related species isadvisable. Alternatively, preparation of an antibody preparation whichexhibits less species specificity may be useful in expression cloningapproaches.

[0045] The phrase “specifically binds to an antibody” or “specificallyimmunoreactive with”, when referring to a protein or peptide, refers toa binding reaction which is determinative of the presence of the proteinin the presence of a heterogeneous population of proteins and otherbiological components. Thus, under designated immunoassay conditions,the specified antibodies bind to a particular protein and do notsignificantly bind other proteins present in the sample. Specificbinding to an antibody under such conditions may require an antibodythat is selected for its specificity for a particular protein. Forexample, antibodies raised to the human CX3Ckine protein immunogen withthe amino acid sequence depicted in SEQ ID NO: 2, 4, 6, or 8 can beselected to obtain antibodies specifically immunoreactive with CX3Ckineproteins and not with other proteins. These antibodies recognizeproteins highly similar to the homologous mouse CX3Ckine protein.

[0046] III. Nucleic Acids

[0047] Human CX3Ckine is exemplary of a larger class of structurally andfunctionally related proteins. These soluble chemokine proteins willserve to transmit signals between different cell types. The preferredembodiments, as disclosed, will be useful in standard procedures toisolate genes from different individuals or other species, e.g., warmblooded animals, such as birds and mammals. Cross hybridization willallow isolation of related genes encoding proteins from individuals,strains, or species. A number of different approaches are available tosuccessfully isolate a suitable nucleic acid clone based upon theinformation provided herein. Southern blot hybridization studies canqualitatively determine the presence of homologous genes in human,monkey, rat, dog, cow, and rabbit genomes under specific hybridizationconditions.

[0048] Complementary sequences will also be used as probes or primers.Based upon identification of the likely amino terminus, other peptidesshould be particularly useful, e.g., coupled with anchored vector orpoly-A complementary PCR techniques or with complementary DNA of otherpeptides. Moreover, reverse translation using the redundancy in thegenetic code may provide synthetic genes which may encode essentiallyidentical proteins often with a condo usage selection preferred forexpression in a given host cell. Techniques for nucleic acidmanipulation of genes encoding CX3Ckine proteins, such as subcloningnucleic acid sequences encoding polypeptides into expression vectors,labelling probes, DNA hybridization, and the like are describedgenerally in Sambrook, et al. (1989) Molecular Cloning: A LaboratoryManual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor Press, N.Y., which is incorporated herein by reference. Thismanual is hereinafter referred to as “Sambrook, et al.” There arevarious methods of isolating DNA sequences encoding CX3Ckine proteins.For example, DNA is isolated from a genomic or cDNA library usinglabeled oligonucleotide probes having sequences identical orcomplementary to the sequences disclosed herein. Full-length probes maybe used, or oligonucleotide probes may be generated by comparison of thesequences disclosed. Such probes can be used directly in hybridizationassays to isolate DNA encoding CX3Ckine proteins, or primers can bedesigned, e.g., using flanking sequence, for use in amplificationtechniques such as PCR, for the isolation of DNA encoding CX3Ckineproteins.

[0049] To prepare a cDNA library, mRNA is isolated from cells whichexpress a CX3Ckine protein. cDNA is prepared from the mRNA and ligatedinto a recombinant vector. The vector is transfected into a recombinanthost for propagation, screening, and cloning. Methods for making andscreening cDNA libraries are well known. See Gubler and Hoffman (1983)Gene 25:263-269 and Sambrook, et al.

[0050] For a genomic library, the DNA can be extracted from tissue andeither mechanically sheared or enzymatically digested to yieldfragments, e.g., of about 12-20 kb. The fragments are then separated bygradient centrifugation and cloned in bacteriophage lambda vectors.These vectors and phage are packaged in vitro, as described in Sambrook,et al. Recombinant phage are analyzed by plaque hybridization asdescribed in Benton and Davis (1977) Science 196:180-182. Colonyhybridization is carried out as generally described in e.g., Grunstein,et al. (1975) Proc. Natl. Acad. Sci. USA. 72:3961-3965.

[0051] DNA encoding a CX3Ckine protein can be identified in either cDNAor genomic libraries by its ability to hybridize with the nucleic acidprobes described herein, e.g., in colony or plaque hybridization assays.The corresponding DNA regions are isolated by standard methods familiarto those of skill in the art. See, e.g., Sambrook, et al. Alternatively,sequence databases, e.g., GenBank, may be evaluated for similar orcorresponding sequences.

[0052] Various methods of amplifying target sequences, such as thepolymerase chain reaction, can also be used to prepare DNA encodingCX3Ckine proteins. Polymerase chain reaction (PCR) technology is used toamplify such nucleic acid sequences directly from mRNA, from cDNA, andfrom genomic libraries or cDNA libraries. The isolated sequencesencoding CX3Ckine proteins may also be used as templates for PCRamplification.

[0053] Typically, in PCR techniques, oligonucleotide primerscomplementary to two 5′ regions in two strands of the DNA region to beamplified are synthesized. The polymerase chain reaction is then carriedout using the two opposite primers. See Innis, et al. (eds.) (1990) PCRProtocols: A Guide to Methods and Applications Academic Press, SanDiego, CA. Primers can be selected to amplify the entire regionsencoding a full-length CX3Ckine protein or to amplify smaller DNAsegments as desired. Once such regions are PCR-amplified, they can besequenced and oligonucleotide probes can be prepared from sequenceobtained using standard techniques. These probes can then be used toisolate DNA's encoding CX3Ckine proteins.

[0054] Oligonucleotides for use as probes are usually chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage and Carruthers (1983) Tetrahedron Lett.22(20):1859-1862, or using an automated synthesizer, as described inNeedham-VanDevanter, et al. (1984) Nucleic Acids Res. 12:6159-6168.Purification of oligonucleotides is performed, e.g., by nativeacrylamide gel electrophoresis or by anion-exchange HPLC as described inPearson and Regnier (1983) J. Chrom. 255:137-149. The sequence of thesynthetic oligonucleotide can be verified using, e.g., the chemicaldegradation method of Maxam, A. M. and Gilbert, W. in Grossman, L. andMoldave (eds.) (1980) Methods in Enzymology 65:499-560 Academic Press,New York.

[0055] An isolated nucleic acid encoding a human CX3Ckine protein wasidentified. The nucleotide sequence and corresponding open reading frameare provided in SEQ ID NO: 1 and 2; with further sequences provided inSEQ ID NO: 3 and 4. Correspondingly, a mouse sequence was identified andits nucleotide and corresponding open reading frame are provided as SEQID NO: 5-8.

[0056] These CX3Ckines exhibit limited similarity to portions ofchemokines, particularly the chemokine domains. See, e.g., Matsushimaand Oppenheim (1989) Cytokine 1:2-13; Oppenheim, et al. (1991) Ann. Rev.Immunol. 9:617-648; Schall (1991) Cytokine 3:165-183; and Gronenborn andClore (1991) Protein Engineering 4:263-269. In particular, the humanCX3Ckine shows similarity to the C class of chemokines in thecarboxyl-terminal portion, particularly with respect to length, and atthe positions corresponding, in the numbering of mature human sequence,to the cys-ala-asp-pro sequence at positions 50-53; and the trp-val atpositions 57-58. CX3Ckines have a much longer carboxyl terminal tailthan the members of the CC or CXC chemokine families, and this “stalk”region may play a role in chemokine presentation. Notably, the spacingof conserved cysteine residues in the CXC and CC families of chemokinesare absent in the human CX3Ckine embodiment. Other features ofcomparison are apparent between the CX3Ckine and chemokine families.See, e.g., Lodi, et al. (1994) Science 263:1762-1766. In particular,β-sheet and α-helix residues can be determined using, e.g., RASMOLprogram, see Sayle and Milner-White (1995) TIBS 20:374-376; orGronenberg, et al. (1991) Protein Engineering 4:263-269; and otherstructural features are defined in Lodi, et al. (1994) Science263:1762-1767. These secondary and tertiary features assist in definingfurther the C, CC, and CXC structural features, along with spacing ofappropriate cysteine residues.

[0057] Based upon the structural modeling and insights in the bindingregions of the chemokines, it is predicted that residues in the maturehuman protein, lacking a signal of 24 residues, 26 (his), 28 (gln), 40(ile), 42 (glu), 47 (arg) and 48 (leu) should be important for chemokinebinding to cells. Residues at the amino terminus are probably notinvolved in receptor binding or specificity.

[0058] Moreover, exon boundaries are predicted to correspond to proteinsegments including the signal sequence through about the second residue(his) in the mature protein; from there to about three residues past thethird cys (around the arg-ala); and from there to the end. The thirdexon appears to exhibit relatively high similarity to the otherchemokines. The second exon would probably be most characteristic of theCX3C chemokines, and would be the preferred segment to use to search forhomology in other variants, e.g, species or otherwise. In particular,segments expected to be preferred in producing CX3C chemokine specificantibodies will include peptides or sequence in the region from thesecond residue of the mature protein (his) through about the thirdresidue after the third cysteine (arg). Fragments of at least about 8-10residues in that region would be especially interesting peptides, e.g.,starting at residue positions of the mature 1, 2, 3, etc. Thosefragments would typically end in that region, e.g., at residue 37, 36,35, etc. Other interesting peptides of various lengths would includeones which begin or end in other positions of the protein, e.g., atresidues 87, 86, etc., with lengths ranging, e.g., from about 8 to 20,25, 30, 35, 40, etc. Corresponding fragments of other mammalainCX3Ckine, e.g., mouse, will be preferred embodiments.

[0059] This invention provides isolated DNA or fragments to encode aCX3Ckine protein. In addition, this invention provides isolated orrecombinant DNA which encodes a protein or polypeptide which is capableof hybridizing under appropriate conditions, e.g., high stringency, withthe DNA sequences described herein. Said biologically active protein orpolypeptide can be an intact ligand, or fragment, and have an amino acidsequence as disclosed in SEQ ID NO: 2, 4, 6, or 8. Preferred embodimentswill be full length natural sequences, from isolates, e.g., about 11,000to 12,500 daltons in size when unglycosylated, or fragments of at leastabout 6,000 daltons, more preferably at least about 8,000 daltons. Inglycosylated form, the protein may exceed 12,500 daltons. Further, thisinvention contemplates the use of isolated or recombinant DNA, orfragments thereof, which encode proteins which are homologous to aCX3Ckine protein or which were isolated using cDNA encoding a CX3Ckineprotein as a probe. The isolated DNA can have the respective regulatorysequences in the 5′ and 3′ flanks, e.g., promoters, enhancers, poly-Aaddition signals, and others.

[0060] IV. Making CX3Ckines

[0061] DNAs which encode a CX3Ckine or fragments thereof can be obtainedby chemical synthesis, screening cDNA libraries, or by screening genomiclibraries prepared from a wide variety of cell lines or tissue samples.The redundancy of the genetic code provides a number of polynucleotidesequences which should encode the same protein.

[0062] These DNAs can be expressed in a wide variety of host cells forthe synthesis of a full-length protein or fragments which can in turn,e.g., be used to generate polyclonal or monoclonal antibodies; forbinding studies; for construction and expression of modified molecules;and for structure/function studies. Each CX3Ckine or its fragments,e.g., the chemokine domain, can be expressed in host cells that aretransformed or transfected with appropriate expression vectors. Thesemolecules can be substantially purified to be free of protein orcellular contaminants, other than those derived from the recombinanthost, and therefore are particularly useful in pharmaceuticalcompositions when combined with a pharmaceutically acceptable carrierand/or diluent. The antigen, e.g., CX3Ckine, or portions thereof, may beexpressed as fusions with other proteins or possessing an epitope tag.Such is applicable also to antigen binding sites.

[0063] Expression vectors are typically self-replicating DNA or RNAconstructs containing the desired antigen gene or its fragments, usuallyoperably linked to appropriate genetic control elements that arerecognized in a suitable host cell. The specific type of controlelements necessary to effect expression will depend upon the eventualhost cell used. Generally, the genetic control elements can include aprokaryotic promoter system or a eukaryotic promoter expression controlsystem, and typically include a transcriptional promoter, an optionaloperator to control the onset of transcription, transcription enhancersto elevate the level of mRNA expression, a sequence that encodes asuitable ribosome binding site, and sequences that terminatetranscription and translation. Expression vectors also usually containan origin of replication that allows the vector to replicateindependently from the host cell.

[0064] The vectors of this invention encompass DNAs which encode aCX3Ckine, or a fragment thereof, typically encoding, e.g., abiologically active polypeptide, or protein. The DNA can be under thecontrol of a viral promoter and can encode a selection marker. Thisinvention further contemplates use of such expression vectors which arecapable of expressing eukaryotic cDNA coding for a CX3Ckine protein in aprokaryotic or eukaryotic host, where the vector is compatible with thehost and where the eukaryotic cDNA coding for the protein is insertedinto the vector such that growth of the host containing the vectorexpresses the cDNA in question. Usually, expression vectors are designedfor stable replication in their host cells or for amplification togreatly increase the total number of copies of the desirable gene percell. It is not always necessary to require that an expression vectorreplicate in a host cell, e.g., it is possible to effect transientexpression of the protein or its fragments in various hosts usingvectors that do not contain a replication origin that is recognized bythe host cell. It is also possible to use vectors that cause integrationof a CX3Ckine gene or its fragments into the host DNA by recombination,or to integrate a promoter which controls expression of an endogenousgene.

[0065] Vectors, as used herein, contemplate plasmids, viruses,bacteriophage, integratable DNA fragments, and other vehicles whichenable the integration of DNA fragments into the genome of the host.Expression vectors are specialized vectors which contain genetic controlelements that effect expression of operably linked genes. Plasmids arethe most commonly used form of vector, but many other forms of vectorswhich serve an equivalent function are suitable for use herein. See,e.g., Pouwels, et al. (1985 and Supplements) Cloning Vectors: ALaboratory Manual Elsevier, N.Y.; and Rodriquez, et al. (eds.) (1988)Vectors: A Survey of Molecular Cloning Vectors and Their UsesButtersworth, Boston, Mass.

[0066] Suitable host cells include prokaryotes, lower eukaryotes, andhigher eukaryotes. Prokaryotes include both gram negative and grampositive organisms, e.g., E. coli and B. subtilis. Lower eukaryotesinclude yeasts, e.g., S. cerevisiae and Pichia, and species of the genusDictyostelium. Higher eukaryotes include established tissue culture celllines from animal cells, both of non-mammalian origin, e.g., insectcells, and birds, and of mammalian origin, e.g., human, primates, androdents.

[0067] Prokaryotic host-vector systems include a wide variety of vectorsfor many different species. As used herein, E. coli and its vectors willbe used generically to include equivalent vectors used in otherprokaryotes. A representative vector for amplifying DNA is pBR322 or itsderivatives. Vectors that can be used to express CX3Ckines or CX3Ckinefragments include, but are not limited to, such vectors as thosecontaining the lac promoter (pUC-series); trp promoter (pBR322-trp); Ipppromoter (the pIN-series); lambda-pP or pR promoters (pOTS); or hybridpromoters such as ptac (pDR540). See Brosius, et al. (1988) “ExpressionVectors Employing Lambda-, trp-, lac-, and Ipp-derived Promoters”, inRodriguez and Denhardt (eds.) Vectors: A Survey of Molecular CloningVectors and Their Uses 10:205-236 Buttersworth, Boston, Mass.

[0068] Lower eukaryotes, e.g., yeasts and Dictyostelium, may betransformed with CX3Ckine sequence containing vectors. For purposes ofthis invention, the most common lower eukaryotic host is the baker'syeast, Saccharomyces cerevisiae. It will be used generically torepresent lower eukaryotes although a number of other strains andspecies are also available. Yeast vectors typically consist of areplication origin (unless of the integrating type), a selection gene, apromoter, DNA encoding the desired protein or its fragments, andsequences for translation termination, polyadenylation, andtranscription termination. Suitable expression vectors for yeast includesuch constitutive promoters as 3-phosphoglycerate kinase and variousother glycolytic enzyme gene promoters or such inducible promoters asthe alcohol dehydrogenase 2 promoter or metallothionine promoter.Suitable vectors include derivatives of the following types:self-replicating low copy number (such as the YRp-series),self-replicating high copy number (such as the YEp-series); integratingtypes (such as the YIp-series), or mini-chromosomes (such as theYCp-series).

[0069] Higher eukaryotic tissue culture cells are typically thepreferred host cells for expression of the functionally active CX3Ckineprotein. In principle, many higher eukaryotic tissue culture cell linesmay be used, e.g., insect baculovirus expression systems, whether froman invertebrate or vertebrate source. However, mammalian cells arepreferred to achieve proper processing, both cotranslationally andposttranslationally. Transformation or transfection and propagation ofsuch cells is routine. Useful cell lines include HeLa cells, Chinesehamster ovary (CHO) cell lines, baby rat kidney (BRK) cell lines, insectcell lines, bird cell lines, and monkey (COS) cell lines. Expressionvectors for such cell lines usually include an origin of replication, apromoter, a translation initiation site, RNA splice sites (e.g., ifgenomic DNA is used), a polyadenylation site, and a transcriptiontermination site. These vectors also may contain a selection gene oramplification gene. Suitable expression vectors may be plasmids,viruses, or retroviruses carrying promoters derived, e.g., from suchsources as from adenovirus, SV40, parvoviruses, vaccinia virus, orcytomegalovirus. Representative examples of suitable expression vectorsinclude pCDNA1; pCD, see Okayama, et al. (1985) Mol. Cell Biol.5:1136-1142; pMClneo Poly-A, see Thomas, et al. (1987) Cell 51:503-512;and a baculovirus vector such as pAC 373 or pAC 610.

[0070] It is likely that CX3Ckines need not be glycosylated to elicitbiological responses. However, it will occasionally be desirable toexpress a CX3Ckine polypeptide in a system which provides a specific ordefined glycosylation pattern. In this case, the usual pattern will bethat provided naturally by the expression system. However, the patternwill be modifiable by exposing the polypeptide, e.g., in unglycosylatedform, to appropriate glycosylating proteins introduced into aheterologous expression system. For example, the CX3Ckine gene may beco-transformed with one or more genes encoding mammalian or otherglycosylating enzymes. It is further understood that over glycosylationmay be detrimental to CX3Ckine biological activity, and that one ofskill may perform routine testing to optimize the degree ofglycosylation which confers optimal biological activity.

[0071] A CX3Ckine, or a fragment thereof, may be engineered to bephosphatidyl inositol (PI) linked to a cell membrane, but can be removedfrom membranes by treatment with a phosphatidyl inositol cleavingenzyme, e.g., phosphatidyl inositol phospholipase-C. This releases theantigen in a biologically active form, and allows purification bystandard procedures of protein chemistry. See, e.g., Low (1989) Biochem.Biophys. Acta 988:427-454; Tse, et al. (1985) Science 230:1003-1008; andBrunner, et al. (1991) J. Cell Biol. 114:1275-1283.

[0072] Now that CX3Ckines have been characterized, fragments orderivatives thereof can be prepared by conventional processes forsynthesizing peptides. These include processes such as are described inStewart and Young (1984) Solid Phase Peptide Synthesis Pierce ChemicalCo., Rockford, Ill.; Bodanszky and Bodanszky (1984) The Practice ofPeptide Synthesis Springer-Verlag, New York, N.Y.; and Bodanszky (1984)The Principles of Peptide Synthesis Springer-Verlag, New York, N.Y. Forexample, an azide process, an acid chloride process, an acid anhydrideprocess, a mixed anhydride process, an active ester process (forexample, p-nitrophenyl ester, N-hydroxysuccinimide ester, or cyanomethylester), a carbodiimidazole process, an oxidative-reductive process, or adicyclohexylcarbodiimide (DCCD)/additive process can be used. Solidphase and solution phase syntheses are both applicable to the foregoingprocesses.

[0073] The prepared protein and fragments thereof can be isolated andpurified from the reaction mixture by means of peptide separation, forexample, by extraction, precipitation, electrophoresis and various formsof chromatography, and the like. The CX3Ckines of this invention can beobtained in varying degrees of purity depending upon its desired use.Purification can be accomplished by use of known protein purificationtechniques or by the use of the antibodies or binding partners hereindescribed, e.g., in immunoabsorbant affinity chromatography. See, e.g.,Coligan, et al. (eds.) (1995 and periodic supplements) Current Protocolsin Protein Science, John Wiley and Sons, New York, N.Y. Thisimmunoabsorbant affinity chromatography is carried out by first linkingthe antibodies to a solid support and then contacting the linkedantibodies with solubilized lysates of appropriate source cells, lysatesof other cells expressing the ligand, or lysates or supernatants ofcells producing the CX3Ckines as a result of recombinant DNA techniques,see below.

[0074] Multiple cell lines may be screened for one which expresses aCX3Ckine at a high level compared with other cells. Various cell lines,e.g., a mouse thymic stromal cell line TA4, is screened and selected forits favorable handling properties. Natural CX3Ckines can be isolatedfrom natural sources, or by expression from a transformed cell using anappropriate expression vector. Purification of the expressed protein isachieved by standard procedures, or may be combined with engineeredmeans for effective purification at high efficiency from cell lysates orsupernatants. Epitope or other tags, e.g., FLAG or His₆ segments, can beused for such purification features.

[0075] V. Antibodies

[0076] Antibodies can be raised to various CX3Ckines, includingindividual, polymorphic, allelic, strain, or species variants, andfragments thereof, both in their naturally occurring (full-length) formsand in their recombinant forms. Additionally, antibodies can be raisedto CX3Ckines in either their active or native forms or in their inactiveor denatured forms. Anti-idiotypic antibodies may also be used.

[0077] A. Antibody Production

[0078] A number of immunogens may be used to produce antibodiesspecifically reactive with CX3Ckine proteins. Recombinant protein is apreferred immunogen for the production of monoclonal or polyclonalantibodies. Naturally occurring protein may also be used either in pureor impure form. Synthetic peptides, made using the human or mouseCX3Ckine protein sequences described herein, may also used as animmunogen for the production of antibodies to CX3Ckines, e.g., thechemokine domains thereof. Recombinant protein can be expressed ineukaryotic or prokaryotic cells as described herein, and purified asdescribed. Naturally folded or denatured material can be used, asappropriate, for producing antibodies. Either monoclonal or polyclonalantibodies may be generated for subsequent use in immunoassays tomeasure the protein.

[0079] Methods of producing polyclonal antibodies are known to those ofskill in the art. Typically, an immunogen, preferably a purifiedprotein, is mixed with an adjuvant and animals are immunized with themixture. The animal's immune response to the immunogen preparation ismonitored by taking test bleeds and determining the titer of reactivityto the CX3Ckine protein or fragment of interest. When appropriately hightiters of antibody to the immunogen are obtained, usually after repeatedimmunizations, blood is collected from the animal and antisera areprepared. Further fractionation of the antisera to enrich for antibodiesreactive to the protein can be done if desired. See, e.g., Harlow andLane; or Coligan.

[0080] Monoclonal antibodies may be obtained by various techniquesfamiliar to those skilled in the art. Typically, spleen cells from ananimal immunized with a desired antigen are immortalized, commonly byfusion with a myeloma cell (see, Kohler and Milstein (1976) Eur. J.Immunol. 6:511-519, incorporated herein by reference). Alternativemethods of immortalization include transformation with Epstein BarrVirus, oncogenes, or retroviruses, or other methods known in the art.Colonies arising from single immortalized cells are screened forproduction of antibodies of the desired specificity and affinity for theantigen, and yield of the monoclonal antibodies produced by such cellsmay be enhanced by various techniques, including injection into theperitoneal cavity of a vertebrate host. Alternatively, one may isolateDNA sequences which encode a monoclonal antibody or a binding fragmentthereof by screening a DNA library from human B cells according, e.g.,to the general protocol outlined by Huse, et al. (1989) Science246:1275-1281.

[0081] Antibodies, including binding fragments and single chainversions, against predetermined fragments of CX3Ckines can be raised byimmunization of animals with conjugates of the fragments with carrierproteins as described above. Monoclonal antibodies are prepared fromcells secreting the desired antibody. These antibodies can be screenedfor binding to normal or defective CX3Ckines, or screened for agonisticor antagonistic activity, e.g., mediated through a receptor. Thesemonoclonal antibodies will usually bind with at least a K_(D) of about 1mM, more usually at least about 300 μM, typically at least about 10 μM,more typically at least about 30 μM, preferably at least about 10 μM,and more preferably at least about 3 μM or better.

[0082] In some instances, it is desirable to prepare monoclonalantibodies from various mammalian hosts, such as mice, rodents,primates, humans, etc. Description of techniques for preparing suchmonoclonal antibodies may be found in, e.g., Stites, et al. (eds.) Basicand Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos,Calif., and references cited therein; Harlow and Lane (1988) Antibodies:A Laboratory Manual CSH Press; Goding (1986) Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press, New York, N.Y.; andparticularly in Kohler and Milstein (1975) Nature 256:495-497, whichdiscusses one method of generating monoclonal antibodies. Summarizedbriefly, this method involves injecting an animal with an immunogen. Theanimal is then sacrificed and cells taken from its spleen, which arethen fused with myeloma cells. The result is a hybrid cell or“hybridoma” that is capable of reproducing in vitro. The population ofhybridomas is then screened to isolate individual clones, each of whichsecrete a single antibody species to the immunogen. In this manner, theindividual antibody species obtained are the products of immortalizedand cloned single B cells from the immune animal generated in responseto a specific site recognized on the immunogenic substance.

[0083] Other suitable techniques involve selection of libraries ofantibodies in phage or similar vectors. See, e.g., Huse, et al. (1989)“Generation of a Large Combinatorial Library of the ImmunoglobulinRepertoire in Phage Lambda,” Science 246:1275-1281; and Ward, et al.(1989) Nature 341:544-546. The polypeptides and antibodies of thepresent invention may be used with or without modification, includingchimeric or humanized antibodies. Frequently, the polypeptides andantibodies will be labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents, teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulinsmay be produced. See, Cabilly, U.S. Pat. No. 4,816,567; and Queen, etal. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033.

[0084] The antibodies of this invention are useful for affinitychromatography in isolating CX3Ckine protein. Columns can be preparedwhere the antibodies are linked to a solid support, e.g., particles,such as agarose, SEPHADEX, or the like, where a cell lysate orsupernatant may be passed through the column, the column washed,followed by increasing concentrations of a mild denaturant, wherebypurified CX3Ckine protein will be released.

[0085] The antibodies may also be used to screen expression librariesfor particular expression products. Usually the antibodies used in sucha procedure will be labeled with a moiety allowing easy detection ofpresence of antigen by antibody binding.

[0086] Antibodies to CX3Ckines may be used for the identification ofcell populations expressing CX3Ckines. By assaying the expressionproducts of cells expressing CX3Ckines it is possible to diagnosedisease, e.g., immune-compromised conditions.

[0087] Antibodies raised against each CX3Ckine will also be useful toraise anti-idiotypic antibodies. These will be useful in detecting ordiagnosing various immunological conditions related to expression of therespective antigens.

[0088] B. Immunoassays

[0089] A particular protein can be measured by a variety of immunoassaymethods. For a review of immunological and immunoassay procedures ingeneral, see Stites and Terr (eds.) (1991) Basic and Clinical Immunology(7th ed.). Moreover, the immunoassays of the present invention can beperformed in many configurations, which are reviewed extensively inMaggio (ed.) (1980) Enzyme Immunoassay CRC Press, Boca Raton, Fla.;Tijan (1985) “Practice and Theory of Enzyme Immunoassays,” LaboratoryTechniques in Biochemistry and Molecular Biology, Elsevier SciencePublishers B.V., Amsterdam; and Harlow and Lane Antibodies A LaboratoryManual, supra, each of which is incorporated herein by reference. Seealso Chan (ed.) (1987) Immunoassay: A Practical Guide Academic Press,Orlando, Fla.; Price and Newman (eds.) (1991) Principles and Practice ofImmunoassays Stockton Press, N.Y.; and Ngo (ed.) (1988) Non-isotopicImmunoassays Plenum Press, N.Y.

[0090] Immunoassays for measurement of CX3Ckine proteins can beperformed by a variety of methods known to those skilled in the art. Inbrief, immunoassays to measure the protein can be competitive ornoncompetitive binding assays. In competitive binding assays, the sampleto be analyzed competes with a labeled analyte for specific bindingsites on a capture agent bound to a solid surface. Preferably thecapture agent is an antibody specifically reactive with CX3Ckineproteins produced as described above. The concentration of labeledanalyte bound to the capture agent is inversely proportional to theamount of free analyte present in the sample.

[0091] In a competitive binding immunoassay, the CX3Ckine proteinpresent in the sample competes with labeled protein for binding to aspecific binding agent, for example, an antibody specifically reactivewith the CX3Ckine protein. The binding agent may be bound to a solidsurface to effect separation of bound labeled protein from the unboundlabeled protein. Alternatively, the competitive binding assay may beconducted in liquid phase and a variety of techniques known in the artmay be used to separate the bound labelled protein from the unboundlabeled protein. Following separation, the amount of bound labeledprotein is determined. The amount of protein present in the sample isinversely proportional to the amount of labeled protein binding.

[0092] Alternatively, a homogeneous immunoassay may be performed inwhich a separation step is not needed. In these immunoassays, the labelon the protein is altered by the binding of the protein to its specificbinding agent. This alteration in the labeled protein results in adecrease or increase in the signal emitted by label, so that measurementof the label at the end of the immunoassay allows for detection orquantitation of the protein.

[0093] CX3Ckine proteins may also be determined by a variety ofnoncompetitive immunoassay methods. For example, a two-site, solid phasesandwich immunoassay may be used. In this type of assay, a binding agentfor the protein, for example an antibody, is attached to a solidsupport. A second protein binding agent, which may also be an antibody,and which binds the protein at a different site, is labelled. Afterbinding at both sites on the protein has occurred, the unbound labeledbinding agent is removed and the amount of labeled binding agent boundto the solid phase is measured. The amount of labeled binding agentbound is directly proportional to the amount of protein in the sample.

[0094] Western blot analysis can be used to determine the presence ofCX3Ckine proteins in a sample. Electrophoresis is carried out, forexample, on a tissue sample suspected of containing the protein.Following electrophoresis to separate the proteins, and transfer of theproteins to a suitable solid support, e.g., a nitrocellulose filter, thesolid support is incubated with an antibody reactive with the protein.This antibody may be labeled, or alternatively may be detected bysubsequent incubation with a second labeled antibody that binds theprimary antibody.

[0095] The immunoassay formats described above employ labeled assaycomponents. The label may be coupled directly or indirectly to thedesired component of the assay according to methods well known in theart. A wide variety of labels and methods may be used. Traditionally, aradioactive label incorporating ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P was used.Non-radioactive labels include ligands which bind to labeled antibodies,fluorophores, chemiluminescent agents, enzymes, and antibodies which canserve as specific binding pair members for a labeled ligand. The choiceof label depends on sensitivity required, ease of conjugation with thecompound, stability requirements, and available instrumentation. For areview of various labelling or signal producing systems which may beused, see U.S. Pat. No. 4,391,904, which is incorporated herein byreference.

[0096] Antibodies reactive with a particular protein can also bemeasured by a variety of immunoassay methods. For a review ofimmunological and immunoassay procedures applicable to the measurementof antibodies by immunoassay techniques, see Stites and Terr (eds.)Basic and Clinical Immunology (7th ed.) supra; Maggio (ed.) EnzymeImmunoassay, supra; and Harlow and Lane Antibodies, A Laboratory Manual,supra.

[0097] In brief, immunoassays to measure antisera reactive with CX3Ckineproteins can be competitive or noncompetitive binding assays. Incompetitive binding assays, the sample analyte competes with a labeledanalyte for specific binding sites on a capture agent bound to a solidsurface. Preferably the capture agent is a purified recombinant CX3Ckineprotein produced as described above. Other sources of CX3Ckine proteins,including isolated or partially purified naturally occurring protein,may also be used. Noncompetitive assays include sandwich assays, inwhich the sample analyte is bound between two analyte-specific bindingreagents. One of the binding agents is used as a capture agent and isbound to a solid surface. The second binding agent is labeled and isused to measure or detect the resultant complex by visual or instrumentmeans. A number of combinations of capture agent and labelled bindingagent can be used. A variety of different immunoassay formats,separation techniques, and labels can be also be used similar to thosedescribed above for the measurement of CX3Ckine proteins.

[0098] VI. Purified CX3Ckines

[0099] Human CX3Ckine amino acid sequences are provided in SEQ ID NO: 2and 4. Mouse nucleotide and amino acid sequences are provided in SEQ IDNO: 5, 6, 7, and 8.

[0100] Purified protein or defined peptides are useful for generatingantibodies by standard methods, as described above. Synthetic peptidesor purified protein, e.g., the chemokine domains, can be presented to animmune system to generate polyclonal and monoclonal antibodies. See,e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY;and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press, NY, which are incorporated herein by reference.Alternatively, a CX3Ckine receptor can be useful as a specific bindingreagent, and advantage can be taken of its specificity of binding, for,e.g., purification of a CX3Ckine ligand.

[0101] The specific binding composition can be used for screening anexpression library made from a cell line which expresses a CX3Ckine.Many methods for screening are available, e.g., standard staining ofsurface expressed ligand, or by panning. Screening of intracellularexpression can also be performed by various staining orimmunofluorescence procedures. The binding compositions could be used toaffinity purify or sort out cells expressing the ligand.

[0102] The peptide segments, along with comparison to homologous genes,can also be used to produce appropriate oligonucleotides to screen alibrary. The genetic code can be used to select appropriateoligonucleotides useful as probes for screening. In combination withpolymerase chain reaction (PCR) techniques, synthetic oligonucleotideswill be useful in selecting desired clones from a library, includingnatural allelic an polymorphic variants.

[0103] The peptide sequences allow preparation of peptides to generateantibodies to recognize such segments, and allow preparation ofoligonucleotides which encode such sequences. The sequence also allowsfor synthetic preparation, e.g., see Dawson, et al. (1994) Science266:776-779. Since CX3Ckines appear to be soluble proteins, the genewill normally possess an N-terminal signal sequence, which is removedupon processing and secretion, and the putative cleavage site is betweenamino acids 24 (gly) and 25 (gln) in SEQ ID NO: 2 or 4, though it may beslightly in either direction. Analysis of the structural features incomparison with the most closely related reported sequences has revealedsimilarities with other cytokines, particularly the class of proteinsknown as chemokines. Within the chemokines are two subgroups, the CC andCXC subgroups. The CX3Ckine family shares various features with each ofthese groups, but its combination of features is distinctive and definesa new family of related chemokines.

[0104] While further structural features result from the sequencesprovided in SEQ ID NO: 1 through 8, the “chemokine on a stick” featureis provided through the stalk region which possesses many sites whichmay provide a heavily glycosylated domain. The stalk structure may beimportant in CX3C chemokine presentation to other cells. In fact, itappears that the stalk region may be processed to release the solublechemokine. This suggests the possibility of substituting the CX3Cchemokine domain with other chemokines to effect efficient presentationto appropriate target cells.

[0105] In addition, the “stalk” regions are likely to affect solubilityand pharmacological aspects of the protein. As such, this region will bethe target of analysis to evaluate and modulate such features aspharmacokenetics. Truncation of that portion may affect half-life,clearance, and acessibility of the chemokine domains.

[0106] VII. Physical Variants

[0107] This invention also encompasses proteins or peptides havingsubstantial amino acid sequence similarity with an amino acid sequenceof a CX3Ckine. Natural variants include individual, polymorphic,allelic, strain, or species variants.

[0108] Amino acid sequence similarity, or sequence identity, isdetermined by optimizing residue matches, if necessary, by introducinggaps as required. This changes when considering conservativesubstitutions as matches. Conservative substitutions typically includesubstitutions within the following groups: glycine, alanine; valine,isoleucine, leucine; aspartic acid, glutamic acid; asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine. Homologous amino acid sequences include natural polymorphic,allelic, and interspecies variations in each respective proteinsequence. Typical homologous proteins or peptides will have from 50-100%similarity (if gaps can be introduced), to 75-100% similarity (ifconservative substitutions are included) with the amino acid sequence ofthe CX3Ckine. Similarity measures will be at least about 50%, generallyat least 60%, more generally at least 65%, usually at least 70%, moreusually at least 75%, preferably at least 80%, and more preferably atleast 80%, and in particularly preferred embodiments, at least 85% ormore. See also Needleham, et al. (1970) J. Mol. Biol. 48:443-453;Sankoff, et al. (1983) Time Warps, String Edits, and Macromolecules: TheTheory and Practice of Sequence Comparison Chapter One, Addison-Wesley,Reading, Mass.; and software packages from IntelliGenetics, MountainView, Calif.; and the University of Wisconsin Genetics Computer Group,Madison, Wis..

[0109] Nucleic acids encoding mammalian CX3Ckine proteins will typicallyhybridize to the nucleic acid sequence of SEQ ID NO: 1, 3, 5 or 7 understringent conditions. For example, nucleic acids encoding human CX3Ckineproteins will normally hybridize to the nucleic acid of SEQ ID NO: 1under stringent hybridization conditions. Generally, stringentconditions are selected to be about 10° C. lower than the thermalmelting point (Tm) for the probe sequence at a defined ionic strengthand pH. The Tm is the temperature (under defined ionic strength and pH)at which 50% of the target sequence hybridizes to a perfectly matchedprobe. Typically, stringent conditions will be those in which the saltconcentration is about 0.2 molar at pH 7 and the temperature is at leastabout 50° C. Other factors may significantly affect the stringency ofhybridization, including, among others, base composition and size of thecomplementary strands, the presence of organic solvents such asformamide, and the extent of base mismatching. A preferred embodimentwill include nucleic acids which will bind to disclosed sequences in 50%formamide and 200 mM NaCl at 42° C.

[0110] An isolated CX3Ckine DNA can be readily modified by nucleotidesubstitutions, nucleotide deletions, nucleotide insertions, and shortinversions of nucleotide stretches. These modifications result in novelDNA sequences which encode CX3Ckine antigens, their derivatives, orproteins having highly similar physiological, immunogenic, or antigenicactivity.

[0111] Modified sequences can be used to produce mutant antigens or toenhance expression. Enhanced expression may involve gene amplification,increased transcription, increased translation, and other mechanisms.Such mutant CX3Ckine derivatives include predetermined or site-specificmutations of the respective protein or its fragments. “Mutant CX3Ckine”encompasses a polypeptide otherwise falling within the homologydefinition of the human CX3Ckine as set forth above, but having an aminoacid sequence which differs from that of a CX3Ckine as found in nature,whether by way of deletion, substitution, or insertion. In particular,“site specific mutant CX3Ckine” generally includes proteins havingsignificant similarity with a protein having a sequence of SEQ ID NO: 2,4, 6, or 8, and as sharing various biological activities, e.g.,antigenic or immunogenic, with those sequences, and in preferredembodiments contain most or all of the disclosed sequence. This appliesalso to polymorphic variants from different individuals. Similarconcepts apply to different CX3Ckine proteins, particularly those foundin various warm blooded animals, e.g., mammals and birds. As statedbefore, it is emphasized that descriptions are generally meant toencompass other CX3Ckine proteins, not limited to the human or mouseembodiments specifically discussed.

[0112] Although site specific mutation sites are predetermined, mutantsneed not be site specific. CX3Ckine mutagenesis can be conducted bymaking amino acid insertions or deletions. Substitutions, deletions,insertions, or any combinations may be generated to arrive at a finalconstruct. These include amino acid residue substitution levels fromnone, one, two, three, five, seven, ten, twelve, fifteen, etc.Insertions include amino- or carboxyl-terminal fusions, e.g. epitopetags. Random mutagenesis can be conducted at a target codon and theexpressed mutants can then be screened for the desired activity. Methodsfor making substitution mutations at predetermined sites in DNA having aknown sequence are well known in the art, e.g., by M13 primermutagenesis or polymerase chain reaction (PCR) techniques. See also,Sambrook, et al. (1989) and Ausubel, et al. (1987 and Supplements). Themutations in the DNA normally should not place coding sequences out ofreading frames and preferably will not create complementary regions thatcould hybridize to produce secondary mRNA structure such as loops orhairpins.

[0113] The present invention also provides recombinant proteins, e.g.,heterologous fusion proteins using segments from these proteins, boththe CX3Ckine, or antigen binding sites. A heterologous fusion protein isa fusion of proteins or segments which are naturally not normally fusedin the same manner. Thus, the fusion product of an immunoglobulin with aCX3Ckine polypeptide is a continuous protein molecule having sequencesfused in a typical peptide linkage, typically made as a singletranslation product and exhibiting properties derived from each sourcepeptide. A similar concept applies to heterologous nucleic acidsequences.

[0114] In addition, new constructs may be made from combining similarfunctional domains from other proteins. For example, protein-binding orother segments may be “swapped” between different new fusionpolypeptides or fragments. See, e.g., Cunningham, et al. (1989) Science243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992.Thus, new chimeric polypeptides exhibiting new combinations ofspecificities will result from the functional linkage of protein-bindingspecificities and other functional domains.

[0115] VIII. Binding Agent:CX3Ckine Protein Complexes

[0116] A CX3Ckine protein that specifically binds to or that isspecifically immunoreactive with an antibody generated against a definedimmunogen, such as an immunogen consisting of the amino acid sequence ofSEQ ID NO: 2. 4, 6, or 8, is typically determined in an immunoassay. Theimmunoassay uses a polyclonal antiserum which was raised to a protein ofSEQ ID NO: 2. 4, 6, or 8. This antiserum is selected to have lowcrossreactivity against other chemokines and any such crossreactivity isremoved by immunoabsorbtion prior to use in the immunoassay.

[0117] In order to produce antisera for use in an immunoassay, theprotein of SEQ ID NO: 2. 4, 6, or 8, is isolated as described herein.For example, recombinant protein may be produced in a mammalian cellline. An inbred strain of mice such as balb/c is immunized with theprotein of SEQ ID NO: 2. 4, 6, or 8, using a standard adjuvant, such asFreund's adjuvant, and a standard mouse immunization protocol (seeHarlow and Lane, supra). Alternatively, a synthetic peptide, preferablynear full length, derived from the sequences disclosed herein andconjugated to a carrier protein can be used an immunogen. Polyclonalsera are collected and titered against the immunogen protein in animmunoassay, for example, a solid phase immunoassay with the immunogenimmobilized on a solid support. Polyclonal antisera with a titer of 10⁴or greater are selected and tested for their cross reactivity against C,C-C, and CXC chemokines, using a competitive binding immunoassay such asthe one described in Harlow and Lane, supra, at pages 570-573.Preferably two chemokines are used in this determination in conjunctionwith either human CX3Ckine or mouse CX3Ckine.

[0118] In conjunction with a CX3Ckine, the monocyte chemotacticprotein-1 (MCP-1) and macrophage inflammatory protein-1α (Mip-1α) areused to identify antibodies which are specifically bound by a CX3Ckine.In conjunction with human CX3Ckine, the monocyte chemotactic protein-2(MCP-2) and Mip-1α are used to identify antibodies which arespecifically bound by a CX3Ckine. These chemokines can be produced asrecombinant proteins and isolated using standard molecular biology andprotein chemistry techniques as described herein.

[0119] Immunoassays in the competitive binding format can be used forthe crossreactivity determinations. For example, a protein of SEQ ID NO:2. 4, 6, or 8 can be immobilized to a solid support. Proteins added tothe assay compete with the binding of the antisera to the immobilizedantigen. The ability of the above proteins to compete with the bindingof the antisera to the immobilized protein is compared to the protein ofSEQ ID NO: 2. 4, 6, or 8. The percent crossreactivity for the aboveproteins is calculated, using standard calculations. Those antisera withless than 10% crossreactivity with each of the proteins listed above areselected and pooled. The cross-reacting antibodies are then removed fromthe pooled antisera by immunoabsorbtion with the above-listed proteins.

[0120] The immunoabsorbed and pooled antisera are then used in acompetitive binding immunoassay as described above to compare a secondprotein to the immunogen protein (e.g., the CX3Ckine chemokine motif ofSEQ ID NO: 2. 4, 6, or 8). In order to make this comparison, the twoproteins are each assayed at a wide range of concentrations and theamount of each protein required to inhibit 50% of the binding of theantisera to the immobilized protein is determined. If the amount of thesecond protein required is less than twice the amount of the protein ofSEQ ID NO: 2 that is required, then the second protein is said tospecifically bind to an antibody generated to the immunogen.

[0121] It is understood that CX3Ckine proteins are a family ofhomologous proteins that comprise two or more genes. For a particulargene product, such as the human CX3Ckine protein, the term refers notonly to the amino acid sequences disclosed herein, but also to otherproteins that are polymorphic, allelic, non-allelic, or speciesvariants. It is also understood that the term “human CX3Ckine” or “mouseCX3Ckine” includes nonnatural mutations introduced by deliberatemutation using conventional recombinant technology such as single sitemutation, or by excising short sections of DNA encoding CX3Ckineproteins, or by substituting new amino acids, or adding new amino acids.Such minor alterations must substantially maintain the immunoidentity ofthe original molecule and/or its biological activity. Thus, thesealterations include proteins that are specifically immunoreactive with adesignated naturally occurring CX3Ckine protein, for example, the humanCX3Ckine protein shown in SEQ ID NO: 2 or 4. The biological propertiesof the altered proteins can be determined by expressing the protein inan appropriate cell line and measuring, e.g., a chemotactic effect.Particular protein modifications considered minor would includeconservative substitution of amino acids with similar chemicalproperties, as described above for the CX3Ckine family as a whole. Byaligning a protein optimally with the protein of SEQ ID NO: 2. 4, 6, or8, and by using the conventional immunoassays described herein todetermine immunoidentity, or by using lymphocyte chemotaxis assays, onecan determine the protein compositions of the invention.

[0122] IX. Functional Variants

[0123] The blocking of physiological response to CX3Ckines may resultfrom the inhibition of binding of the protein to its receptor, e.g.,through competitive inhibition. Thus, in vitro assays of the presentinvention will often use isolated protein, membranes from cellsexpressing a recombinant membrane associated CX3Ckine, soluble fragmentscomprising receptor binding segments of these proteins, or fragmentsattached to solid phase substrates. These assays will also allow for thediagnostic determination of the effects of either binding segmentmutations and modifications, or protein mutations and modifications,e.g., protein analogs. This invention also contemplates the use ofcompetitive drug screening assays, e.g., where neutralizing antibodiesto antigen or receptor fragments compete with a test compound forbinding to the protein. In this manner, the antibodies can be used todetect the presence of a polypeptide which shares one or more antigenicbinding sites of the protein and can also be used to occupy bindingsites on the protein that might otherwise interact with a receptor.

[0124] “Derivatives” of CX3Ckine antigens include amino acid sequencemutants, glycosylation variants, and covalent or aggregate conjugateswith other chemical moieties. Covalent derivatives can be prepared bylinkage of functionalities to groups which are found in CX3Ckine aminoacid side chains or at the N- or C-termini, by means which are wellknown in the art. These derivatives can include, without limitation,aliphatic esters or amides of the carboxyl terminus, or of residuescontaining carboxyl side chains, O-acyl derivatives of hydroxylgroup-containing residues, and N-acyl derivatives of the amino terminalamino acid or amino-group containing residues, e.g., lysine or arginine.Acyl groups are selected from the group of alkyl-moieties including C3to C18 normal alkyl, thereby forming alkanoyl aroyl species. See,e.g.,Coligan, et al. (eds.) (1995 and periodic supplements) Current Protocolsin Protein Science, John Wiley and Sons, New York, NY. Covalentattachment to carrier proteins may be important when immunogenicmoieties are haptens.

[0125] In particular, glycosylation alterations are included, e.g., madeby modifying the glycosylation patterns of a polypeptide during itssynthesis and processing, or in further processing steps. Particularlypreferred means for accomplishing this are by exposing the polypeptideto glycosylating enzymes derived from cells which normally provide suchprocessing, e.g., mammalian glycosylation enzymes. Deglycosylationenzymes are also contemplated. Also embraced are versions of the sameprimary amino acid sequence which have other minor modifications,including phosphorylated amino acid residues, e.g., phosphotyrosine,phosphoserine, or phosphothreonine, or other moieties, including ribosylgroups or cross-linking reagents.

[0126] A major group of derivatives are covalent conjugates of theCX3Ckine or fragments thereof with other proteins or polypeptides. Thesederivatives can be synthesized in recombinant culture such as N- orC-terminal fusions or by the use of agents known in the art for theirusefulness in cross-linking proteins through reactive side groups.Preferred protein derivatization sites with cross-linking agents are atfree amino groups, carbohydrate moieties, and cysteine residues.

[0127] Fusion polypeptides between CX3Ckines and other homologous orheterologous proteins are also provided. Many growth factors andcytokines are homodimeric entities, and a repeat construct may havevarious advantages, including lessened susceptibility to proteolyticdegradation. Moreover, many receptors require dimerization to transducea signal, and various dimeric proteins or domain repeats can bedesirable. Heterologous polypeptides may be fusions between differentsurface markers, resulting in, e.g., a hybrid protein exhibitingreceptor binding specificity. Likewise, heterologous fusions may beconstructed which would exhibit a combination of properties oractivities of the derivative proteins. Typical examples are fusions of areporter polypeptide, e.g., luciferase, with a segment or domain of aprotein, e.g., a receptor-binding segment, so that the presence orlocation of the fused protein may be easily determined. See, e.g., Dull,et al., U.S. Pat. No. 4,859,609. Other gene fusion partners includebacterial β-galactosidase, trpE, Protein A, β-lactamase, alpha amylase,alcohol dehydrogenase, and yeast alpha mating factor. See, e.g., See,e.g., Dawson, et al. (1994) Science 266:776-779; and Godowski, et al.(1988) Science 241:812-816. In particular, fusion proteins with portionsfrom the related genes will be useful. Similar concepts of fusions withantigen binding sites are contemplated.

[0128] Such polypeptides may also have amino acid residues which havebeen chemically modified by phosphorylation, sulfonation, biotinylation,or the addition or removal of other moieties, particularly those whichhave molecular shapes similar to phosphate groups. In some embodiments,the modifications will be useful labeling reagents, or serve aspurification targets, e.g., affinity ligands.

[0129] This invention also contemplates the use of derivatives ofCX3Ckines other than variations in amino acid sequence or glycosylation.Such derivatives may involve covalent or aggregative association withchemical moieties. These derivatives include: (1) salts, (2) side chainand terminal residue covalent modifications, and (3) adsorptioncomplexes, for example with cell membranes. Such covalent or aggregativederivatives are useful as immunogens, as reagents in immunoassays, or inpurification methods such as for affinity purification of ligands orother binding ligands. For example, a CX3Ckine antigen can beimmobilized by covalent bonding to a solid support such as cyanogenbromide-activated SEPHAROSE, by methods which are well known in the art,or adsorbed onto polyolefin surfaces, with or without glutaraldehydecross-linking, for use in the assay or purification of anti-CX3Ckineantibodies or its receptor. The CX3Ckines can also be labeled with adetectable group, e.g., radioiodinated by the chloramine T procedure,covalently bound to rare earth chelates, or conjugated to anotherfluorescent moiety for use in diagnostic assays. Purification ofCX3Ckines may be effected by immobilized antibodies or receptor.

[0130] Isolated CX3Ckine genes will allow transformation of cellslacking expression of corresponding CX3Ckines, e.g., either speciestypes or cells which lack corresponding proteins and exhibit negativebackground activity. Expression of transformed genes will allowisolation of antigenically pure cell lines, with defined or singlespecie variants. This approach will allow for more sensitive detectionand discrimination of the physiological effects of CX3Ckine receptorproteins. Subcellular fragments, e.g., cytoplasts or membrane fragments,can be isolated and used.

[0131] X. Uses

[0132] The present invention provides reagents which will find use indiagnostic applications as described elsewhere herein, e.g., in thegeneral description for developmental abnormalities, or below in thedescription of kits for diagnosis.

[0133] CX3Ckine nucleotides, e.g., human or mouse CX3Ckine DNA or RNA,may be used as a component in a forensic assay. For instance, thenucleotide sequences provided may be labeled using, e.g., ³²P or biotinand used to probe standard restriction fragment polymorphism blots,providing a measurable character to aid in distinguishing betweenindividuals. Such probes may be used in well-known forensic techniquessuch as genetic fingerprinting. In addition, nucleotide probes made fromCX3Ckine sequences may be used in in situ assays to detect chromosomalabnormalities. For instance, rearrangements in the mouse chromosomeencoding a CX3Ckine gene may be detected via well-known in situtechniques, using CX3Ckine probes in conjunction with other knownchromosome markers.

[0134] Antibodies and other binding agents directed towards CX3Ckineproteins or nucleic acids may be used to purify the correspondingCX3Ckine molecule. As described in the Examples below, antibodypurification of CX3Ckine components is both possible and practicable.Antibodies and other binding agents may also be used in a diagnosticfashion to determine whether CX3Ckine components are present in a tissuesample or cell population using well-known techniques described herein.The ability to attach a binding agent to a CX3Ckine provides a means todiagnose disorders associated with CX3Ckine misregulation. Antibodiesand other CX3Ckine binding agents may also be useful as histologicalmarkers. As described in the examples below, CX3Ckine expression islimited to specific tissue types. By directing a probe, such as anantibody or nucleic acid to a CX3Ckine it is possible to use the probeto distinguish tissue and cell types in situ or in vitro.

[0135] This invention also provides reagents with significanttherapeutic value. The CX3Ckines (naturally occurring or recombinant),fragments thereof, and antibodies thereto, along with compoundsidentified as having binding affinity to a CX3Ckine, are useful in thetreatment of conditions associated with abnormal physiology ordevelopment, including abnormal proliferation, e.g., cancerousconditions, or degenerative conditions. Abnormal proliferation,regeneration, degeneration, and atrophy may be modulated by appropriatetherapeutic treatment using the compositions provided herein. Forexample, a disease or disorder associated with abnormal expression orabnormal signaling by a CX3Ckine is a target for an agonist orantagonist of the protein. The proteins likely play a role in regulationor development of neuronal or hematopoietic cells, e.g., lymphoid cells,which affect immunological responses.

[0136] Other abnormal developmental conditions are known in cell typesshown to possess CX3Ckine mRNA by northern blot analysis. See Berkow(ed.) The Merck Manual of Diagnosis and Therapy, Merck & Co., Rahway,N.J.; and Thorn, et al. Harrison's Principles of Internal Medicine,McGraw-Hill, N.Y. Developmental or functional abnormalities, e.g., ofthe neuronal or immune system, cause significant medical abnormalitiesand conditions which may be susceptible to prevention or treatment usingcompositions provided herein.

[0137] Certain chemokines have also been implicated in viral replicationmechanisms. See, e.g., Cohen (1996) Science 272:809-810; Feng, et al.(1996) Science 272:872-877; and Cocchi, et al. (1995) Science270:1811-1816. The CX3C chemokine may be useful in a similar context.Alternatively, the stalk structure may be very important in presentationof the ligand domain, and other chemokines may be advantageouslysubstituted for the chemokine domain in this molecule. Modification inthe “stalk” structure may affect many of the pharmacological propertiesof the CX3Ckine, including half-life and biological activity.

[0138] Recombinant CX3Ckine or CX3Ckine antibodies can be purified andthen administered to a patient, e.g., in sterile form. These reagentscan be combined for therapeutic use with additional active or inertingredients, e.g., in conventional pharmaceutically acceptable carriersor diluents, e.g., immunogenic adjuvants, along with physiologicallyinnocuous stabilizers and excipients. These combinations can be sterilefiltered and placed into dosage forms as by lyophilization in dosagevials or storage in stabilized aqueous preparations. This invention alsocontemplates use of antibodies or binding fragments thereof, includingforms which are not complement binding.

[0139] Drug screening using antibodies or receptor or fragments thereofcan identify compounds having binding affinity to CX3Ckines, includingisolation of associated components. Subsequent biological assays canthen be utilized to determine if the compound has intrinsic stimulatingactivity and is therefore a blocker or antagonist in that it blocks theactivity of the protein. Likewise, a compound having intrinsicstimulating activity can activate the receptor and is thus an agonist inthat it simulates the activity of a CX3Ckine. This invention furthercontemplates the therapeutic use of antibodies to CX3Ckines asantagonists. This approach should be particularly useful with otherCX3Ckine species variants.

[0140] The quantities of reagents necessary for effective therapy willdepend upon many different factors, including means of administration,target site, physiological state of the patient, and other medicantsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in situ administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described, e.g., in Gilman, et al. (eds.)(1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics(8th ed.) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences(17th ed.) Mack Publishing Co., Easton, Pa. Methods for administrationare discussed therein and below, e.g., for oral, intravenous,intraperitoneal, or intramuscular administration, transdermal diffusion,and others. Pharmaceutically acceptable carriers will include water,saline, buffers, and other compounds described, e.g., in the MerckIndex, Merck & Co., Rahway, N.J. Dosage ranges would ordinarily beexpected to be in amounts lower than 1 mM concentrations, typically lessthan about 10 μM concentrations, usually less than about 100 nM,preferably less than about 10 pM (picomolar), and most preferably lessthan about 1 fM (femtomolar), with an appropriate carrier. Slow releaseformulations, or a slow release apparatus will often be utilized forcontinuous administration.

[0141] CX3Ckines, fragments thereof, and antibodies to it or itsfragments, antagonists, and agonists, may be administered directly tothe host to be treated or, depending on the size of the compounds, itmay be desirable to conjugate them to carrier proteins such as ovalbuminor serum albumin prior to their administration. Therapeutic formulationsmay be administered in many conventional dosage formulations. While itis possible for the active ingredient to be administered alone, it ispreferable to present it as a pharmaceutical formulation. Formulationstypically comprise at least one active ingredient, as defined above,together with one or more acceptable carriers thereof. Each carriershould be both pharmaceutically and physiologically acceptable in thesense of being compatible with the other ingredients and not injuriousto the patient. Formulations include those suitable for oral, rectal,nasal, or parenteral (including subcutaneous, intramuscular, intravenousand intradermal) administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. See, e.g., Gilman, et al. (eds.) (1990)Goodman and Gilman's: The Pharmacological Bases of Therapeutics (8thed.) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences(17th ed.) Mack Publishing Co., Easton, Pa.; Avis, et al. (eds.) (1993)Pharmaceutical Dosage Forms: Parenteral Medications Dekker, N.Y.;Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: TabletsDekker, N.Y.; and Lieberman, et al. (eds.) (1990) Pharmaceutical DosageForms: Disperse Systems Dekker, N.Y. The therapy of this invention maybe combined with or used in association with other therapeutic agents.

[0142] Both the naturally occurring and the recombinant forms of theCX3Ckines of this invention are particularly useful in kits and assaymethods which are capable of screening compounds for binding activity tothe proteins. Several methods of automating assays have been developedin recent years so as to permit screening of tens of thousands ofcompounds in a short period. See, e.g., Fodor, et al. (1991) Science251:767-773, and other descriptions of chemical diversity libraries,which describe means for testing of binding affinity by a plurality ofcompounds. The development of suitable assays can be greatly facilitatedby the availability of large amounts of purified, soluble CX3Ckine asprovided by this invention.

[0143] For example, antagonists can normally be found once the proteinhas been structurally defined. Testing of potential protein analogs isnow possible upon the development of highly automated assay methodsusing a purified receptor. In particular, new agonists and antagonistswill be discovered by using screening techniques described herein. Ofparticular importance are compounds found to have a combined bindingaffinity for multiple CX3Ckine receptors, e.g., compounds which canserve as antagonists for species variants of a CX3Ckine.

[0144] This invention is particularly useful for screening compounds byusing recombinant protein in a variety of drug screening techniques. Theadvantages of using a recombinant protein in screening for specificligands include: (a) improved renewable source of the CX3Ckine from aspecific source; (b) potentially greater number of ligands per cellgiving better signal to noise ratio in assays; and (c) species variantspecificity (theoretically giving greater biological and diseasespecificity).

[0145] One method of drug screening utilizes eukaryotic or prokaryotichost cells which are stably transformed with recombinant DNA moleculesexpressing a CX3Ckine receptor. Cells may be isolated which express areceptor in isolation from any others. Such cells, either in viable orfixed form, can be used for standard ligand/receptor binding assays. Seealso, Parce, et al. (1989) Science 246:243-247; and Owicki, et al.(1990) Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitivemethods to detect cellular responses. Competitive assays areparticularly useful, where the cells (source of CX3Ckine) are contactedand incubated with a labeled receptor or antibody having known bindingaffinity to the ligand, such as ¹²⁵I-antibody, and a test sample whosebinding affinity to the binding composition is being measured. The boundand free labeled binding compositions are then separated to assess thedegree of ligand binding. The amount of test compound bound is inverselyproportional to the amount of labeled receptor binding to the knownsource. Any one of numerous techniques can be used to separate boundfrom free ligand to assess the degree of ligand binding. This separationstep could typically involve a procedure such as adhesion to filtersfollowed by washing, adhesion to plastic followed by washing, orcentrifugation of the cell membranes. Viable cells could also be used toscreen for the effects of drugs on CX3Ckine mediated functions, e.g.,second messenger levels, i.e., Ca⁺⁺; cell proliferation; inositolphosphate pool changes; and others. Some detection methods allow forelimination of a separation step, e.g., a proximity sensitive detectionsystem. Calcium sensitive dyes will be useful for detecting Ca⁺⁺ levels,with a fluorimeter or a fluorescence cell sorting apparatus.

[0146] Another method utilizes membranes from transformed eukaryotic orprokaryotic host cells as the source of a CX3Ckine. These cells arestably transformed with DNA vectors directing the expression of aCX3Ckine, e.g., an engineered membrane bound form. Essentially, themembranes would be prepared from the cells and used in a receptor/ligandbinding assay such as the competitive assay set forth above.

[0147] Still another approach is to use solubilized, unpurified orsolubilized, purified CX3Ckine from transformed eukaryotic orprokaryotic host cells. This allows for a “molecular” binding assay withthe advantages of increased specificity, the ability to automate, andhigh drug test throughput.

[0148] Another technique for drug screening involves an approach whichprovides high throughput screening for compounds having suitable bindingaffinity to a CX3Ckine antibody and is described in detail in Geysen,European Patent Application 84/03564, published on Sep. 13, 1984. First,large numbers of different small peptide test compounds are synthesizedon a solid substrate, e.g., plastic pins or some other appropriatesurface, see Fodor, et al., supra. Then all the pins are reacted withsolubilized, unpurified or solubilized, purified CX3Ckine antibody, andwashed. The next step involves detecting bound CX3Ckine antibody.

[0149] Rational drug design may also be based upon structural studies ofthe molecular shapes of the CX3Ckine and other effectors or analogs.See, e.g., Methods in Enzymology vols 202 and 203. Effectors may beother proteins which mediate other functions in response to ligandbinding, or other proteins which normally interact with the receptor.One means for determining which sites interact with specific otherproteins is a physical structure determination, e.g., x-raycrystallography or 2 dimensional NMR techniques. These will provideguidance as to which amino acid residues form molecular contact regions.For a detailed description of protein structural determination, see,e.g., Blundell and Johnson (1976) Protein Crystallography AcademicPress, NY.

[0150] A purified CX3Ckine can be coated directly onto plates for use inthe aforementioned drug screening techniques. However, non-neutralizingantibodies to these ligands can be used as capture antibodies toimmobilize the respective ligand on the solid phase.

[0151] XI. Kits

[0152] This invention also contemplates use of CX3Ckine proteins,fragments thereof, peptides, and their fusion products in a variety ofdiagnostic kits and methods for detecting the presence of CX3Ckine or aCX3Ckine receptor. Typically the kit will have a compartment containingeither a defined CX3Ckine peptide or gene segment or a reagent whichrecognizes one or the other, e.g., receptor fragments or antibodies.

[0153] A kit for determining the binding affinity of a test compound toa CX3Ckine would typically comprise a test compound; a labeled compound,e.g., a receptor or antibody having known binding affinity for theCX3Ckine; a source of CX3Ckine (naturally occurring or recombinant); anda means for separating bound from free labeled compound, such as a solidphase for immobilizing the CX3Ckine. Once compounds are screened, thosehaving suitable binding affinity to the CX3Ckine can be evaluated insuitable biological assays, as are well known in the art, to determinewhether they act as agonists or antagonists to the receptor. Theavailability of recombinant CX3Ckine polypeptides also provide welldefined standards for calibrating such assays.

[0154] A preferred kit for determining the concentration of, forexample, a CX3Ckine in a sample would typically comprise a labeledcompound, e.g., receptor or antibody, having known binding affinity forthe CX3Ckine, a source of CX3Ckine (naturally occurring or recombinant),and a means for separating the bound from free labeled compound, forexample, a solid phase for immobilizing the CX3Ckine. Compartmentscontaining reagents, and instructions, will normally be provided.

[0155] Antibodies, including antigen binding fragments, specific for theCX3Ckine or ligand fragments are useful in diagnostic applications todetect the presence of elevated levels of CX3Ckine and/or its fragments.Such may allow diagnosis of the amounts of differently processed formsof the CX3Ckine, e.g., successively degraded stalk structure. Suchdiagnostic assays can employ lysates, live cells, fixed cells,immunofluorescence, cell cultures, body fluids, and further can involvethe detection of antigens related to the ligand in serum, or the like.Diagnostic assays may be homogeneous (without a separation step betweenfree reagent and antigen-CX3Ckine complex) or heterogeneous (with aseparation step). Various commercial assays exist, such asradioimmunoassay (RIA), enzyme-linked immunosorbentassay (ELISA), enzymeimmunoassay (EIA), enzyme-multiplied immunoassay technique (EMIT),substrate-labeled fluorescent immunoassay (SLFIA), and the like. Forexample, unlabeled antibodies can be employed by using a second antibodywhich is labeled and which recognizes the antibody to a CX3Ckine or to aparticular fragment thereof. Similar assays have also been extensivelydiscussed in the literature. See, e.g., Harlow and Lane (1988)Antibodies: A Laboratory Manual, CSH Press, NY; Chan (ed.) (1987)Immunoassay: A Practical Guide Academic Press, Orlando, FL; Price andNewman (eds.) (1991) Principles and Practice of Immunoassay StocktonPress, NY; and Ngo (ed.) (1988) Nonisotopic Immunoassay Plenum Press,NY.

[0156] Anti-idiotypic antibodies may have similar use to diagnosepresence of antibodies against a CX3Ckine, as such may be diagnostic ofvarious abnormal states. For example, overproduction of CX3Ckine mayresult in production of various immunological or other medical reactionswhich may be diagnostic of abnormal physiological states, e.g., in cellgrowth, acitivation, or differentiation.

[0157] Frequently, the reagents for diagnostic assays are supplied inkits, so as to optimize the sensitivity of the assay. For the subjectinvention, depending upon the nature of the assay, the protocol, and thelabel, either labeled or unlabeled antibody or receptor, or labeledCX3Ckine is provided. This is usually in conjunction with otheradditives, such as buffers, stabilizers, materials necessary for signalproduction such as substrates for enzymes, and the like. Preferably, thekit will also contain instructions for proper use and disposal of thecontents after use. Typically the kit has compartments for each usefulreagent. Desirably, the reagents are provided as a dry lyophilizedpowder, where the reagents may be reconstituted in an aqueous mediumproviding appropriate concentrations of reagents for performing theassay.

[0158] Many of the aforementioned constituents of the drug screening andthe diagnostic assays may be used without modification, or may bemodified in a variety of ways. For example, labeling may be achieved bycovalently or non-covalently joining a moiety which directly orindirectly provides a detectable signal. In any of these assays, theprotein, test compound, CX3Ckine, or antibodies thereto can be labeledeither directly or indirectly. Possibilities for direct labeling includelabel groups: radiolabels such as ¹²⁵I, enzymes (U.S. Pat. No.3,645,090) such as peroxidase and alkaline phosphatase, and fluorescentlabels (U.S. Pat. No. 3,940,475) capable of monitoring the change influorescence intensity, wavelength shift, or fluorescence polarization.Possibilities for indirect labeling include biotinylation of oneconstituent followed by binding to avidin coupled to one of the abovelabel groups.

[0159] There are also numerous methods of separating the bound from thefree ligand, or alternatively the bound from the free test compound. TheCX3Ckine can be immobilized on various matrices followed by washing.Suitable matrices include plastic such as an ELISA plate, filters, andbeads. Methods of immobilizing the CX3Ckine to a matrix include, withoutlimitation, direct adhesion to plastic, use of a capture antibody,chemical coupling, and biotin-avidin. The last step in this approachinvolves the precipitation of ligand/receptor or ligand/antibody complexby any of several methods including those utilizing, e.g., an organicsolvent such as polyethylene glycol or a salt such as ammonium sulfate.Other suitable separation techniques include, without limitation, thefluorescein antibody magnetizable particle method described in Rattle,et al. (1984) Clin. Chem. 30:1457-1461, and the double antibody magneticparticle separation as described in U.S. Pat. No. 4,659,678.

[0160] Methods for linking proteins or their fragments to the variouslabels have been extensively reported in the literature and do notrequire detailed discussion here. Many of the techniques involve the useof activated carboxyl groups either through the use of carbodiimide oractive esters to form peptide bonds, the formation of thioethers byreaction of a mercapto group with an activated halogen such aschloroacetyl, or an activated olefin such as maleimide, for linkage, orthe like. Fusion proteins will also find use in these applications.

[0161] Another diagnostic aspect of this invention involves use ofoligonucleotide or polynucleotide sequences taken from the sequence of aCX3Ckine. These sequences can be used as probes for detecting levels ofthe CX3Ckine message in samples from natural sources, or patientssuspected of having an abnormal condition, e.g., cancer or developmentalproblem. The preparation of both RNA and DNA nucleotide sequences, thelabeling of the sequences, and the preferred. size of the sequences hasreceived ample description and discussion in the literature. Normally anoligonucleotide probe should have at least about 14 nucleotides, usuallyat least about 18 nucleotides, and the polynucleotide probes may be upto several kilobases. Various labels may be employed, most commonlyradionuclides, particularly ³²P. However, other techniques may also beemployed, such as using biotin modified nucleotides for introductioninto a polynucleotide. The biotin then serves as the site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionuclides, fluorophores, enzymes, or the like.Alternatively, antibodies may be employed which can recognize specificduplexes, including DNA duplexes, RNA duplexes, DNA-RNA hybrid duplexes,or DNA-protein duplexes. The antibodies in turn may be labeled and theassay carried out where the duplex is bound to a surface, so that uponthe formation of duplex on the surface, the presence of antibody boundto the duplex can be detected. The use of probes to the novel anti-senseRNA may be carried out using many conventional techniques such asnucleic acid hybridization, plus and minus screening, recombinationalprobing, hybrid released translation (HRT), and hybrid arrestedtranslation (HART). This also includes amplification techniques such aspolymerase chain reaction (PCR).

[0162] Diagnostic kits which also test for the qualitative orquantitative presence of other markers are also contemplated. Diagnosisor prognosis may depend on the combination of multiple indications usedas markers. Thus, kits may test for combinations of markers. See, e.g.,Viallet, et al. (1989) Progress in Growth Factor Res. 1:89-97.

[0163] XII. Receptor Isolation

[0164] Having isolated a binding partner of a specific interaction,methods exist for isolating the counter-partner. See, Gearing, et al.(1989) EMBO J. 8:3667-3676. For example, means to label a CX3Ckinewithout interfering with the binding to its receptor can be determined.For example, an affinity label or epitope tag can be fused to either theamino- or carboxyl-terminus of the ligand. An expression library can bescreened for specific binding of the CX3Ckine, e.g., by cell sorting, orother screening to detect subpopulations which express such a bindingcomponent. See, e.g., Ho, et al. (1993) Proc. Nat'l Acad. Sci. USA90:11267-11271. Alternatively, a panning method may be used. See, e.g.,Seed and Aruffo (1987) Proc. Nat'l Acad. Sci. USA 84:3365-3369. Atwo-hybrid slection system may also be applied making appropriateconstructs with the available BAS-1 sequences. See, e.g., Fields andSong (1989) Nature 340:245-246.

[0165] Protein cross-linking techniques with label can be applied toisolate binding partners of a CX3Ckine. This would allow identificationof proteins which specifically interact with a CX3Ckine, e.g., in aligand-receptor like manner. Typically, the chemokine family binds toreceptors of the seven transmembrane receptor family, and the receptorfor the CX3Ckine is likely to exhibit a similar structure. Thus, it islikely that the receptor will be found by expression in a system whichis capable of expressing such a membrane protein in a form capable ofexhibiting ligand binding capability.

[0166] The broad scope of this invention is best understood withreference to the following examples, which are not intended to limit theinvention to specific embodiments.

EXAMPLES

[0167] I. General Methods

[0168] Many of the standard methods below are described or referenced,e.g., in Maniatis, et al. (1982) Molecular Cloning, A Laboratory ManualCold Spring Harbor Laboratory, Cold Spring Harbor Press, NY; Sambrook,et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.) Vols. 1-3,CSH Press, NY; Ausubel, et al., Biology Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements) CurrentProtocols in Molecular Biology Wiley/Greene, NY; Innis, et al. (eds.)(1990) PCR Protocols: A Guide to Methods and Applications AcademicPress, NY. Methods for protein purification include such methods asammonium sulfate precipitation, column chromatography, electrophoresis,centrifugation, crystallization, and others. See, e.g., Ausubel, et al.(1987 and periodic supplements); Deutscher (1990) “Guide to ProteinPurification,” Methods in Enzymology vol. 182, and other volumes in thisseries; and manufacturer's literature on use of protein purificationproducts, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond,Calif. Combination with recombinant techniques allow fusion toappropriate segments (epitope tags), e.g., to a FLAG sequence or anequivalent which can be fused, e.g., via a protease-removable sequence.See, e.g., Hochuli (1989) Chemische Industrie 12:69-70; Hochuli (1990)“Purification of Recombinant Proteins with Metal Chelate Absorbent” inSetlow (ed.) Genetic Engineering, Principle and Methods 12:87-98, PlenumPress, NY; Crowe, et al. (1992) QIAexDress: The High Level Expression &Protein Purification System QIAGEN, Inc., Chatsworth, Calif.; andColigan, et al. (eds.) (1995 and periodic supplements) Current Protocolsin Protein Science, John Wiley and Sons, New York, N.Y.

[0169] Standard immunological techniques are described, e.g., in Coligan(1991) Current Protocols in Immunology Wiley/Greene, NY; and Methods inEnzymology volumes. 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150,162, and 163. Assays for neural cell biological activities aredescribed, e.g., in Wouterlood (ed. 1995) Neuroscience Protocols modules10, Elsevier; Methods in Neurosciences Academic Press; and NeuromethodsHumana Press, Totowa, N.J. Methodology of developmental systems isdescribed, e.g., in Meisami (ed.) Handbook of Human Growth andDevelopmental Biology CRC Press; and Chrispeels (ed.) MolecularTechniques and Approaches in Developmental Biology Interscience.

[0170] FACS analyses are described in Melamed, et al. (1990) FlowCytometry and Sorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988)Practical Flow Cytometry Liss, New York, N.Y.; and Robinson, et al.(1993) Handbook of Flow Cytometry Methods Wiley-Liss, New York, N.Y.

[0171] II. Isolation of Human CX3Ckine Clone

[0172] A clone encoding the human CX3Ckine is isolated from a naturalsource by many different possible methods. Given the sequences providedherein, PCR primers or hybridization probes are selected and/orconstructed to isolate either genomic DNA segments or cDNA reversetranscripts. Appropriate cell sources include human tissues, e.g., brainlibraries. Tissue distribution below also suggests source tissues.Genetic and polymorphic or allelic variants are isolated by screening apopulation of individuals.

[0173] PCR based detection is performed by standard methods, preferablyusing primers from opposite ends of the coding sequence, but flankingsegments might be selected for specific purposes.

[0174] Alternatively, hybridization probes are selected. Particular ATor GC contents of probes are selected depending upon the expectedhomology and mismatching expected. Appropriate stringency conditions areselected to balance an appropriate positive signal to background ratio.Successive washing steps are used to collect clones of greater homology.

[0175] Further clones are isolated using an antibody based selectionprocedure. Standard expression cloning methods are applied including,e.g., FACS staining of membrane associated expression product. Theantibodies are used to identify clones producing a recognized protein.Alternatively, antibodies are used to purify a CX3C chemokine, withprotein sequencing and standard means to isolate a gene encoding thatprotein.

[0176] Genomic sequence based methods will also allow for identificationof sequences naturally available, or otherwise, which exhibit homologyto the provided sequences. Similar procedures will allow isolation ofother primate genes.

[0177] III. Isolation of Rodent CX3Ckine Clone

[0178] Similar methods are used as above to isolate an appropriate mouseCX3C chemokine gene. Similar source materials as indicated above areused to isolate natural genes, including genetic, polymorphic, allelic,or strain variants. Species variants are also isolated using similarmethods, e.g., from rats, moles, muskrats, copybaras, etc.

[0179] IV. Isolation of an Avian CX3Ckine Clone

[0180] An appropriate avian source is selected as above. Similar methodsare utilized to isolate a species variant, though the level ofsimilarity will typically be lower for avian CX3C chemokine as comparedto a human to mouse sequence.

[0181] V. Expression; Purification; Characterization

[0182] With an appropriate clone from above, the coding sequence isinserted into an appropriate expression vector. This may be in a vectorspecifically selected for a prokaryote, yeast, insect, or highervertebrate, e.g., mammalian expression system. Standard methods areapplied to produce the gene product, preferably as a soluble secretedmolecule, but will, in certain instances, also be made as anintracellular protein. Intracellular proteins typically require celllysis to recover the protein, and insoluble inclusion bodies are acommon starting material for further purificiation.

[0183] With a clone encoding a vertebrate CX3C chemokine, recombinantproduction means are used, although natural forms may be purified fromappropriate sources. The protein product is purified by standard methodsof protein purification, in certain cases, e.g., coupled withimmunoaffinity methods. Immunoaffinity methods are used either as apurification step, as described above, or as a detection assay todetermine the separation properties of the protein.

[0184] Preferably, the protein is secreted into the medium, and thesoluble product is purified from the medium in a soluble form.Alternatively, as described above, inclusion bodies from prokaryoticexpression systems are a useful source of material. Typically, theinsoluble protein is solubilized from the inclusion bodies and refoldedusing standard methods. Purification methods are developed as describedabove.

[0185] In certain embodiments, the protein is made in a eukaryotic cellwhich glycosylates the protein normally. The purification methods may beaffected thereby, as may biological activities. The intact protein canbe processed to release the chemokine domain, probably due to a proteasecleavage event somewhere in the glycosylated stalk region close to thechemokine/stalk boundary. While recombinant protein appears to beprocessed, the physiological processes which normally do such in nativecells remain to be determined.

[0186] The product of the purification method described above ischaracterized to determine many structural features. Standard physicalmethods are applied, e.g., amino acid analysis and protein sequencing.The resulting protein is subjected to CD spectroscopy and otherspectroscopic methods, e.g., NMR, ESR, mass spectroscopy, etc. Theproduct is characterized to determine its molecular form and size, e.g.,using gel chromatography and similar techniques. Understanding of thechromatographic properties will lead to more gentle or efficientpurification methods.

[0187] CX3C chemokine protein biochemistry was assessed in mammalianexpression systems. Human embryonic kidney 293 cells (HEK 293)transfected with a mammalian expression construct encoding full-lengthCX3C chemokine were metabolically labeled with ³⁵S cysteine andmethionine. CX3C chemokine was produced as a protein of Mr ˜95 kDa;control transfected supernatants contained no such species.Neuraminidase and gycosidases reduced the Mr of CX3C chemokine from ˜95kDa to ˜45 kDa, suggesting that the recombinant form is glycosylatedsubstantially. Thus CX3C chemokine cDNA, encoding a predictedmembrane-bound protein, encodes a glycoprotein which is released fromcells by an undefined mechanism.

[0188] Prediction of glycosylation sites may be made, e.g., as reportedin Hansen, et al. (1995) Biochem. J. 308:801-813.

[0189] VI. Preparation of Antibodies Against Vertebrate CX3Ckine

[0190] With protein produce, as above, animals are immunized to produceantibodies. Polyclonal antiserum is raised using non-purified antigen,though the resulting serum will exhibit higher background levels.Preferably, the antigen is purified using standard protein purificationtechniques, including, e.g., affinity chromatography using polyclonalserum indicated above. Presence of specific antibodies is detected usingdefined synthetic peptide fragments. Preferred fragements include thechemokine domain.

[0191] Polyclonal serum is raised against a purified antigen, purifiedas indicated above, or using synthetic peptides. A series of overlappingsynthetic peptides which encompass all of the full length sequence, ifpresented to an animal, will produce serum recognizing most linearepitopes on the protein. Such an antiserum is used to affinity purifyprotein, which is, in turn, used to introduce intact full length proteininto another animal to produce another antiserum preparation.

[0192] Similar techniques are used to generate induce monoclonalantibodies to either unpurified antigen, or, preferably, purifiedantigen.

[0193] VII. Cellular and Tissue Distribution

[0194] Distribution of the protein or gene products are determined,e.g., using immunohistochemistry with an antibody reagent, as producedabove, or by screening for nucleic acids encoding the chemokine. Eitherhybridization or PCR methods are used to detect DNA, cDNA, or messagecontent. Histochemistry allows determination of the specific cell typeswithin a tissue which express higher or lower levels of message or DNA.Antibody techniques are useful to quantitate protein in a biologicalsample, including a liquid or tissue sample. Immunoassays are developedto quantitate protein.

[0195] Hybridization techniques were applied to the tissue types inTable 3 with positive or negative results, as indicated. The commercialtissue blots may have cellular contamination from resident cells, e.g.,from blood or other cells which populate the tissue. The large and smalltranscripts correspond to sizes about 4 kb and less than about 2 kb,respectively. TABLE 3 Tissue and cell distribution of human CX3Ckinegene. Commercial tissue library: cell type large small spleen − −thymus + − prostate + + testis + − ovary + − small intestine + +colan + + peripheral blood − −

[0196] Further analysis of tissue distribution indicates abundance ofhuman message: heart +++; brain +++; placenta −; lung ++; liver −;muscle +; kidney −; pancreas +; spleen −; thymus +; prostate ++; testis+; ovary +; small intestine ++; colon ++; peripheral blood −; HL60promyelocytic leukemia line −; HeLa cell S3 −; K562 chronic myelogenousleukemia line −; Molt4 lymphoblastic leukemia line −; Burkitts lymphomaRAJI line ; SW480 colorectal adenocarcinoma line +; A549 lung carcinomaline −; and G361 melanoma line −.

[0197] “Reverse northerns” are blots from cDNA libraries with theinserts removed, and the size determinations are based upon the size ofinserts in the cDNA library, and reflect the lengths found in the cDNAlibrary inserts, which may be less than full length where the reversetranscription was not full length. As such, size determinations thereare not reflective of the natural sizes. The results of these are: PBMC(peripheral blood mononuclear cells) +; PBMC (activated using T cellstimulation conditions, with anti-CD3 and PMA) −; Mot72 (resting ThOclone) +; Mot 72 (activated with anti-CD28 and anti-CD3) −; Mot72 α(activated with anti-peptide, anergic clone) −; Mot81 (resting Th0clone) −; Mot81 (activated with anti-CD28 and anti-CD3) −; HY06 (restingTh1 clone) −; HY06 (activated with anti-CD28 and anti-CD3) −; HY06α(activated with anti-peptide, anergic clone) −; HY935 (resting Th2clone) −; HY935 (activated with anti-CD28 and anti-CD3) +; BC pool ofEBV transformed lines +; resting splenocytes +; splenocytes +(activatedusing B cell stimulating conditions, with anti-CD40 and IL-4) −; NK cellpool −; NK pool (activated 6 h with PMA and ionomycin) +; NKA6 NK cellclone −; NKB1 NK cell clone −; NK non-cytotoxic cell clone +; and NKclone stimulated to be cytotoxic −. Other cells and tissues: CHO cells+; Jurkat cells (DNAX) +; Jurkat cells (another source) +; normal T cellpool +; TCT pool (transformed T cells) −; fetal kidney −; fetal lung −;fetal liver −; fetal heart −; fetal brain +; fetal gall bladder +; fetalsmall intestine +; fetal adipose +; fetal ovary −; fetal uterus +; adultplacenta −; fetal testis +; fetal spleen +; and fetal brain +.Additional cells provided: U937 (resting monocyte cell line) +; C−(elutriated monocyte activated with LPS, IFN-γ, and anti-IL-10) +; C+(elutriated monocytes activated with LPS, IFN-γ, and IL-10) +; M1(elutriated monocytes activated with LPS 1 h) +; M6 (elutriatedmonocytes activated with LPS 6 h) +; 30% DC (resting 30% CD1a+dendriticcells, proliferated in TNF-α and GM-CSF) +; 70% DC (resting 70%CD1a+dendritic cells, proliferated in TNF-α and GM-CSF) +; D1 (dendriticcells stimulated 1 h in PMA and ionomycin) −; D6 (dendritic cellsstimulated 6 h in PMA and ionomycin) −; D5 DC (resting dendritic cellscultured 5 d in GM-CSF and IL-4) +; DC (dendritic cells cultured inGM-CSF and IL-4, LPS activated) +; DC (GM-CSF activated, like D5 cells)+; DC mix (dendritic cells stimulated with a mixture of cytokines) +;CD1a+(99% pure CD1a+dendritic cells, enriched from 70% DC) +;CD14+(CD14+fraction sorted from 70% DC, monocyte-like morphology) −;CD1Aa+(95% CD1a+and CD86+sorted from 70% DC) −; TF1 (hematopoieticprecursor line) +; Jurkat (T cell line) +; MRC5 (lung fibroblast sarcomacell line) +; JY (B cell line) +; U937 (pre-monocytic cell line) +.

[0198] Since the endothelium is a major site of chemokine action, anorthern blot was performed to ascertain if CX3Ckine was expressed inthis tissue. Human CX3Ckine was also shown to be expressed on humanactivated primary endothelial cells by both mRNA and protein expression.This suggests that CX3Ckine may be involved in leukocyte trafficking invarious organs.

[0199] In summary, human CX3Ckine mRNA is found in monocytes, dendriticcells, T cells and B cells, e.g., found in certain immune cells.

[0200] VIII. Microchemotaxis Assays

[0201] The pro-migratory activities of CX3C chemokine have been assessedin microchemotaxis assays. See, e.g., Bacon, et al. (1988) Br. J.Pharmacol. 95:966-974. CX3C chemokine appears to be a potent attractantof peripheral blood monocytes and T cells. Pro-migratory activity forblood neutrophils has been difficult to demonstrate.

[0202] IX. Chromosomal Mapping

[0203] The CX3C chemokine gene has been mapped to human chromosome 16. ABIOS Laboratories (New Haven, Conn.) mouse somatic cell hybrid panel wascombined with PCR. These mapping studies also indicate the possibilityof a pseudogene or related gene on human chromosome 14. Sequencing ofgenomic DNA fragments suggests CX3C chemokine gene has an intron whichbegins near or in the codon encoding Ile 64. Other intron/exonboundaries have yet to be mapped. This location is distinct from thechromosomal mapping locations of the other C, CC, or CXC chemokinefamilies, consistent with the CX3Ckine being a separate gene familywithin the chemokines.

[0204] X. Biological Activities, Direct and Indirect

[0205] The 293 human embryonic kidney cell line (HEK 293) wastransfected with either the membrane bound form of human CX3Ckine(293-CX3Ckine), the chemokine domain plus the “stalk” region, or acontrol vector without an insert. The transfected cells weresubsequently cultured with either monocytes, T cells, or peripheralmononuclear (PMN) cells to assay relative adherence of these cells toCX3Ckine. Specifically, 5×10⁴ cells per well of HEK 293 transfectedcells were seeded in a 96 well plate. 2×10⁵ monocytes, T cells, or PMNs,metabolically labeled with ³⁵S-methionine and cysteine (Amersham,Arlington Heights, Ill.), were added to each well. The plate was thenincubated at 37° C. for varius time points. The wells were washed 2times RPMI supplemented with 1% FCS. Plates were then read in aMillipore Cytofluor at 485/530 nm.

[0206] In all cases, adherence to HEK 293 cells transfected with themembrane bound form of CX3Ckine was signifigantly enhanced when comparedto the truncated CX3Ckine or mock transfected cells. Interestingly, onlythe membrane bound form possessed this proadhesive activity, leading tothe conclusion that CX3Ckine, in its membrane bound form, may serve as aregulator of circulating leukocytes.

[0207] In another experiment, the recombinant soluble form of thechemokine domain of CX3Ckine (rCx3C) was added to HEK 293-CX3C cells andmonocytes at a concentration of 1 μM per well, and assayed as decribedabove. rCX3C was able to antagonize adhesion of monocytes to HEK293-CX3C cells. A similar experiment was performed to investigate theeffect on T cell adherence. Comparable results were obtained. Thus rCX3Cmay function as a negative regulator of circulating leukocytes.

[0208] A comparison of three different forms of human CX3Ckine wasperformed to analyze variations in chemoattractant activity that may bedue to the structure of CX3Ckine. CX3C 1.7 (chemokine domain plus theentire stalk region), CX3C 0.7 (chemokine domain plus one-half stalkregion), and CX3C CK (chemokine domain only) were subjected to thechemotaxicity assay described above, their ability to attract T cellswas analyzed. CX3C 1.7 displayed a slightly better dose dependentability to attract T cells relative to the other forms of CX3Ckine.

[0209] A robust and sensitive assay is selected as described above,e.g., on immune cells, neuronal cells, or stem cells. Chemokine is addedto the assay in increasing doses to see if a dose response is detected.For example, in a proliferation assay, cells are plated out in plates.Appropriate culture medium is provided, and chemokine is added to thecells in varying amounts. Growth is monitored over a period of timewhich will detect either a direct effect on the cells, or an indirecteffect of the chemokine.

[0210] Alternatively, an activation assay or attraction assay. is used.An appropriate cell type is selected, e.g, hematopoietic cells, myeloid(macrophages, neutrophils, polymorphonuclear cells, etc.) or lymphoid (Tcell, B cell, or NK cells), neural cells (neurons, neuroglia,oligodendrocytes, astrocytes, etc.), or stem cells, e.g., progenitorcells which differentiate to other cell types, e.g., gut crypt cells andundifferentiated cell types.

[0211] Other assays will be those which have been demonstrated withother chemokines. See, e.g., Schall and Bacon (1994) Current Opinion inImmunology 6:865-873; and Bacon and Schall (1996) Int. Arch. Allergy &Immunol. 109:97-109. Effects of truncated stalk structures will besimilarly evaluated.

[0212] XI. Structure Activity Relationship

[0213] Information on the criticality of particular residues isdetermined using standard procedures and analysis. Standard mutagenesisanalysis is performed, e.g., by generating many different variants atdetermined positions, e.g., at the positions identified above, andevaluating biological activities of the variants. This may be performedto the extent of determining positions which modify activity, or tofocus on specific positions to determine the residues which can besubstituted to either retain, block, or modulate biological activity.

[0214] Alternatively, analysis of natural variants can indicate whatpositions tolerate natural mutations. This may result from populationalanalysis of variation among individuals, or across strains or species.Samples from selected individuals are analysed, e.g., by PCR analysisand sequencing. This allows evaluation of population polymorphisms.Particularly, as described above, many of the biological activities ofthe chemokine domain attached to different portions or extents of thestalk structure may result.

[0215] XII. Screening for Agonists/Antagonists

[0216] Agonists or antagonists are screened for ability to induce orblock biological activity. The candidate compounds, e.g, sequencevariants of natural CX3Ckines, are assayed for their biologiclaactivities. Alternatively, compounds are screened, alone or incombinations, to determine effects on biological activity.

[0217] XIII. Isolation of a Receptor for CX3C Chemokine

[0218] Based on the proadherent properties of CX3Ckine, 7 transmembraneG-protein receptor was found to be expressed by monocytes and T cells.It was also discovered that the chemokine domain is the only region ofCX3Ckine that can engage the receptor. Binding assays with knownchemokine receptor revealed that CX3Ckine does not engage chemokinereceptors CCR 1 through 5, CXCR 1 and 2, or the Duffy antigen receptor.CX3Ckine can, however, bind to a virally encoded chemokine receptor,CMV-US28.

[0219] Alternatively, CX3C chemokine can be used as a specific bindingreagent to identify its binding partner, by taking advantage of itsspecificity of binding, much like an antibody would be used. A bindingreagent is either labeled as described above, e.g., fluorescence orotherwise, or immobilized to a substrate for panning methods. Thetypical chemokine receptor is a seven transmembrane receptor.

[0220] The purified protein is also be used to identify other bindingpartners of CX3Ckine as described, e.g., in Fields and Song (1989)Nature 340:245-246.

[0221] The binding composition, e.g., chemokine, is used to screen anexpression library made from a cell line which expresses a bindingpartner, i.e. receptor. Standard staining techniques are used to detector sort intracellular or surface expressed receptor, or surfaceexpressing transformed cells are screened by panning. Screening ofintracellular expression is performed by various staining orimmunofluorescence procedures. See also McMahan, et al. (1991) EMBO J.10:2821-2832.

[0222] For example, on day 0, precoat 2-chamber permanox slides with 1ml per chamber of fibronectin, 10 ng/ml in PBS, for 30 min at roomtemperature. Rinse once with PBS. Then plate COS cells at 2-3×10⁵ cellsper chamber in 1.5 ml of growth media. Incubate overnight at 37° C.

[0223] On day 1 for each sample, prepare 0.5 ml of a solution of 66μg/ml DEAE-dextran, 66 μM chloroquine, and 4 μg DNA in serum free DME.For each set, a positive control is prepared, e.g., of human CX3Cchemokine cDNA at 1 and {fraction (1/200)} dilution, and a negativemock. Rinse cells with serum free DME. Add the DNA solution and incubate5 hr at 37° C. Remove the medium and add 0.5 ml 10% DMSO in DME for 2.5min. Remove and wash once with DME. Add 1.5 ml growth medium andincubate overnight.

[0224] On day 2, change the medium. On days 3 or 4, the cells are fixedand stained. Rinse the cells twice with Hank's Buffered Saline Solution(HBSS) and fix in 4% paraformaldehyde (PFA)/glucose for 5 min. Wash 3×with HBSS. The slides may be stored at −80° C. after all liquid isremoved. For each chamber, 0.5 ml incubations are performed as follows.Add HBSS/saponin (0.1%) with 32 μl/ml of 1 M NaN₃ for 20 min. Cells arethen washed with HBSS/saponin 1×. Add chemokine or chemokine/antibodycomplex to cells and incubate for 30 min. Wash cells twice withHBSS/saponin. If appropriate, add first antibody for 30 min. Add secondantibody, e.g., Vector anti-mouse antibody, at {fraction (1/200)}dilution, and incubate for 30 min. Prepare ELISA solution, e.g., VectorElite ABC horseradish peroxidase solution, and preincubate for 30 min.Use, e.g., 1 drop of solution A (avidin) and 1 drop solution B (biotin)per 2.5 ml HBSS/saponin. Wash cells twice with HBSS/saponin. Add ABC HRPsolution and incubate for 30 min. Wash cells twice with HBSS, secondwash for 2 min, which closes cells. Then add Vector diaminobenzoic acid(DAB) for 5 to 10 min. Use 2 drops of buffer plus 4 drops DAB plus 2drops of H₂O₂ per 5 ml of glass distilled water. Carefully removechamber and rinse slide in water. Air dry for a few minutes, then add 1drop of Crystal Mount and a cover slip. Bake for 5 min at 85-90° C.

[0225] Evaluate positive staining of pools and pregressively subclone toisolation of single genes responsible for the binding.

[0226] Alternatively, chemokine reagents are used to affinity purify orsort out cells expressing a receptor. See, e.g., Sambrook, et al. orAusubel, et al.

[0227] Another strategy is to screen for a membrane bound receptor bypanning. The receptor cDNA is constructed as described above. The ligandcan be immobilized and used to immobilize expressing cells.Immobilization may be achieved by use of appropriate antibodies whichrecognize, e.g., a FLAG sequence of a chemokine fusion construct, or byuse of antibodies raised against the first antibodies. Recursive cyclesof selection and amplification lead to enrichment of appropriate clonesand eventual isolation of receptor expressing clones.

[0228] Phage expression libraries can be screened by chemokine.Appropriate label techniques, e.g., anti-FLAG antibodies, will allowspecific labeling of appropriate clones.

[0229] All references cited herein are incorporated herein by referenceto the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference in its entirety for all purposes.

[0230] Many modifications and variations of this invention can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1 11 534 base pairs nucleic acid single linear cDNA CDS 89..424 1CCCAAGCTTG GCACGAGGGC ACTGAGCTCT GCCGCCTGGC TCTAGCCGCC TGCCTGGCCC 60CCGCCGGGAC TCTTGCCCAC CCTCAGCCAT GGCTCCGATA TCTCTGTCGT GGCTGCTCCG 120CTTGGCCACC TTCTGCCATC TGACTGTCCT GCTGGCTGGA CAGCACCACG GTGTGACGAA 180ATGCAACATC ACGTGCAGCA AGATGACATC AAAGATACCT GTAGCTTTGC TCATCCACTA 240TCAACAGAAC CAGGCATCAT GCGGCAAACG CGCAATCATC TTGGAGACGA GACAGCACAG 300GCTGTTCTGT GCCGACCCGA AGGAGCAATG GGTCAAGGAC GCGATGCAGC ATCTGGACCG 360CCAGGCTGCT GCCCTAACTC CGAAATGGCG GCACCTTCCG AAGAAGCCAG ATCGGCGAGG 420TTGAAGCCCA GGACCACCCC CTGCCGCCGG GGGAAATGGA CNAGTCTGTT GGTCCCTGGA 480ACCCCGAAAG CCCACAGGCG AAAAGCCAGT TACCCTGGAN CCGAATCCTT CTTC 534 111amino acids amino acid single linear peptide 2 Met Ala Pro Ile Ser LeuSer Trp Leu Leu Arg Leu Ala Thr Phe Cys 1 5 10 15 His Leu Thr Val LeuLeu Ala Gly Gln His His Gly Val Thr Lys Cys 20 25 30 Asn Ile Thr Cys SerLys Met Thr Ser Lys Ile Pro Val Ala Leu Leu 35 40 45 Ile His Tyr Gln GlnAsn Gln Ala Ser Cys Gly Lys Arg Ala Ile Ile 50 55 60 Leu Glu Thr Arg GlnHis Arg Leu Phe Cys Ala Asp Pro Lys Glu Gln 65 70 75 80 Trp Val Lys AspAla Met Gln His Leu Asp Arg Gln Ala Ala Ala Leu 85 90 95 Thr Pro Lys TrpArg His Leu Pro Lys Lys Pro Asp Arg Arg Gly 100 105 110 1654 base pairsnucleic acid single linear cDNA CDS 86..1279 3 AAGCTTGGCA CGAGGGCACTGAGCTCTGCC GCCTGGCTCT AGCCGCCTGC CTGGCCCCCG 60 CCGGGACTCT TGCCCACCCTCAGCCATGGC TCCGATATCT CTGTCGTGGC TGCTCCGCTT 120 GGCCACCTTC TGCCATCTGACTGTCCTGCT GGCTGGACAG CACCACGGTG TGACGAAATG 180 CAACATCACG TGCAGCAAGATGACATCAAA GATACCTGTA GCTTTGCTCA TCCACTATCA 240 ACAGAACCAG GCATCATGCGGCAAACGCGC AATCATCTTG GAGACGAGAC AGCACAGGCT 300 GTTCTGTGCC GACCCGAAGGAGCAATGGGT CAAGGACGCG ATGCAGCATC TGGACCGCCA 360 GGCTGCTGCC CTAACTCGAAATGGCGGCAC CTTCGAGAAG CAGATCGGCG AGGTGAAGCC 420 CAGGACCACC CCTGCCGCCGGGGGAATGGA CGAGTCTGTG GTCCTGGAGC CCGAAGCCAC 480 AGGCGAAAGC AGTAGCCTGGAGCCGACTCC TTCTTCCCAG GAAGCACAGA GGGCCCTGGG 540 GACCTCCCCA GAGCTGCCGACGGGCGTGAC TGGTTCCTCA GGGACCAGGC TCCCCCCGAC 600 GCCAAAGGCT CAGGATGGAGGGCCTGTGGG CACGGAGCTT TTCCGAGTGC CTCCCGTCTC 660 CACTGCCGCC ACGTGGCAGAGTTCTGCTCC CCACCAACCT GGGCCCAGCC TCTGGGCTGA 720 GGCAAAGACC TCTGAGGCCCCGTCCACCCA GGACCCCTCC ACCCAGGCCT CCACTGCGTC 780 CTCCCCAGCC CCAGAGGAGAATGCTCCGTC TGAAGGCCAG CGTGTGTGGG GTCAGGGACA 840 GAGCCCCAGG CCAGAGAACTCTCTGGAGCG GGAGGAGATG GGTCCCGTGC CAGCGCACAC 900 GGATGCCTTC CAGGACTGGGGGCCTGGCAG CATGGCCCAC GTCTCTGTGG TCCCTGTCTC 960 CTCAGAAGGG ACCCCCAGCAGGGAGCCAGT GGCTTCAGGC AGCTGGACCC CTAAGGCTGA 1020 GGAACCCATC CATGCCACCATGGACCCCCA GAGGCTGGGC GTCCTTATCA CTCCTGTCCC 1080 TGACGCCCAG GCTGCCACCCGGAGGCAGGC GGTGGGGCTG CTGGCCTTCC TTGGCCTCCT 1140 CTTCTGCCTG GGGGTGGCCATGTTCACCTA CCAGAGCCTC CAGGGCTGCC CTCGAAAGAT 1200 GGCAGGAGAG ATGGCGGAGGGCCTTCGCTA CATCCCCCGG AGCTGTGGTA GTAATTCATA 1260 TGTCCTGGTG CCCGTGTGAACTCCTCTGGC CTGTGTCTAG TTGTTTGATT CAGACAGCTG 1320 CCTGGGATCC CTCATCCTCATACCCACCCC CACCCAAGGG CCTGGCCTGA GCTGGGATGA 1380 TTGGAGGGGG GAGGTGGGATCCTCCAGGTG CACAAGCTCC AAGCTCCCAG GCATTCCCCA 1440 GGAGGCCAGC CTTGACCATTCTCCACCTTC CAGGGACAGA GGGGGTGGCC TCCCAACTCA 1500 CCCCAGCCCC AAAACTCTCCTCTGCTGCTG GCTGGTTAGA GGTTCCCTTT GACGCCATCC 1560 CAGCCCCAAT GAACAATTATTTATTAAATG CCCAGCCCCT TCTGAAAAAA AAAAAAAAAA 1620 AAAAAAAAAA AAAAAAAAAAATTCCTGCGG CCGC 1654 397 amino acids amino acid single linear peptide 4Met Ala Pro Ile Ser Leu Ser Trp Leu Leu Arg Leu Ala Thr Phe Cys 1 5 1015 His Leu Thr Val Leu Leu Ala Gly Gln His His Gly Val Thr Lys Cys 20 2530 Asn Ile Thr Cys Ser Lys Met Thr Ser Lys Ile Pro Val Ala Leu Leu 35 4045 Ile His Tyr Gln Gln Asn Gln Ala Ser Cys Gly Lys Arg Ala Ile Ile 50 5560 Leu Glu Thr Arg Gln His Arg Leu Phe Cys Ala Asp Pro Lys Glu Gln 65 7075 80 Trp Val Lys Asp Ala Met Gln His Leu Asp Arg Gln Ala Ala Ala Leu 8590 95 Thr Arg Asn Gly Gly Thr Phe Glu Lys Gln Ile Gly Glu Val Lys Pro100 105 110 Arg Thr Thr Pro Ala Ala Gly Gly Met Asp Glu Ser Val Val LeuGlu 115 120 125 Pro Glu Ala Thr Gly Glu Ser Ser Ser Leu Glu Pro Thr ProSer Ser 130 135 140 Gln Glu Ala Gln Arg Ala Leu Gly Thr Ser Pro Glu LeuPro Thr Gly 145 150 155 160 Val Thr Gly Ser Ser Gly Thr Arg Leu Pro ProThr Pro Lys Ala Gln 165 170 175 Asp Gly Gly Pro Val Gly Thr Glu Leu PheArg Val Pro Pro Val Ser 180 185 190 Thr Ala Ala Thr Trp Gln Ser Ser AlaPro His Gln Pro Gly Pro Ser 195 200 205 Leu Trp Ala Glu Ala Lys Thr SerGlu Ala Pro Ser Thr Gln Asp Pro 210 215 220 Ser Thr Gln Ala Ser Thr AlaSer Ser Pro Ala Pro Glu Glu Asn Ala 225 230 235 240 Pro Ser Glu Gly GlnArg Val Trp Gly Gln Gly Gln Ser Pro Arg Pro 245 250 255 Glu Asn Ser LeuGlu Arg Glu Glu Met Gly Pro Val Pro Ala His Thr 260 265 270 Asp Ala PheGln Asp Trp Gly Pro Gly Ser Met Ala His Val Ser Val 275 280 285 Val ProVal Ser Ser Glu Gly Thr Pro Ser Arg Glu Pro Val Ala Ser 290 295 300 GlySer Trp Thr Pro Lys Ala Glu Glu Pro Ile His Ala Thr Met Asp 305 310 315320 Pro Gln Arg Leu Gly Val Leu Ile Thr Pro Val Pro Asp Ala Gln Ala 325330 335 Ala Thr Arg Arg Gln Ala Val Gly Leu Leu Ala Phe Leu Gly Leu Leu340 345 350 Phe Cys Leu Gly Val Ala Met Phe Thr Tyr Gln Ser Leu Gln GlyCys 355 360 365 Pro Arg Lys Met Ala Gly Glu Met Ala Glu Gly Leu Arg TyrIle Pro 370 375 380 Arg Ser Cys Gly Ser Asn Ser Tyr Val Leu Val Pro Val385 390 395 209 base pairs nucleic acid single linear cDNA CDS 63..209 5TNACTACTAG GAGCTGCGAC ACGGCCCAGC CTCCTGGCCC GNCGAATTCC TGCACTCCAG 60CCATGGCTCC CTCGCCGCTC GCGTGGCTGC TGCGCCTGGC CGCGTTCTTC CATTTGTGTA 120CTCTGCTGCC GGGTNAGCAC CTCGGCATGA CGAAATGCGA AATCATGTGC GACAAGATGA 180CCTNACGAAT NCCAGTGGCT TTANTCATC 209 49 amino acids amino acid singlelinear peptide 6 Met Ala Pro Ser Pro Leu Ala Trp Leu Leu Arg Leu Ala AlaPhe Phe 1 5 10 15 His Leu Cys Thr Leu Leu Pro Gly Xaa His Leu Gly MetThr Lys Cys 20 25 30 Glu Ile Met Cys Asp Lys Met Thr Xaa Arg Xaa Pro ValAla Leu Xaa 35 40 45 Ile 3065 base pairs nucleic acid single linear cDNACDS 62..1249 7 TGACTACTAG GAGCTGCGAC ACGGCCCAGC CTCCTGGCCG CCGAATTCCTGCACTCCAGC 60 C ATG GCT CCC TCG CCG CTC GCG TGG CTG CTG CGC CTG GCC GCGTTC 106 Met Ala Pro Ser Pro Leu Ala Trp Leu Leu Arg Leu Ala Ala Phe 1 510 15 TTC CAT TTG TGT ACT CTG CTG CCG GGT CAG CAC CTC GGC ATG ACG AAA154 Phe His Leu Cys Thr Leu Leu Pro Gly Gln His Leu Gly Met Thr Lys 2025 30 TGC GAA ATC ATG TGC GGC AAG ATG ACC TCA CGA ATC CCA GTG GCT TTG202 Cys Glu Ile Met Cys Gly Lys Met Thr Ser Arg Ile Pro Val Ala Leu 3540 45 CTC ATC CGC TAT CAG CTA AAT CAG GAG TCC TGC GGC AAG CGT GCC ATT250 Leu Ile Arg Tyr Gln Leu Asn Gln Glu Ser Cys Gly Lys Arg Ala Ile 5055 60 GTC CTG GAG ACG ACA CAG CAC AGA CGC TTC TGT GCT GAC CCG AAG GAG298 Val Leu Glu Thr Thr Gln His Arg Arg Phe Cys Ala Asp Pro Lys Glu 6570 75 AAA TGG GTC CAA GAC GCC ATG AAG CAT CTG GAT CAC CAG GCT GCT GCC346 Lys Trp Val Gln Asp Ala Met Lys His Leu Asp His Gln Ala Ala Ala 8085 90 95 CTC ACT AAA AAT GGT GGC AAG TTT GAG AAG CGG GTG GAC AAT GTG ACA394 Leu Thr Lys Asn Gly Gly Lys Phe Glu Lys Arg Val Asp Asn Val Thr 100105 110 CCT GGG ATC ACC TTG GCC ACT AGG GGA CTG TCC CCA TCT GCC CTG ACA442 Pro Gly Ile Thr Leu Ala Thr Arg Gly Leu Ser Pro Ser Ala Leu Thr 115120 125 AAG CCT GAA TCC GCC ACA TTG GAA GAC CTT GCT TTG GAA CTG ACT ACT490 Lys Pro Glu Ser Ala Thr Leu Glu Asp Leu Ala Leu Glu Leu Thr Thr 130135 140 ATT TCC CAG GAG GCC AGG GGG ACC ATG GGG ACT TCC CAA GAG CCA CCG538 Ile Ser Gln Glu Ala Arg Gly Thr Met Gly Thr Ser Gln Glu Pro Pro 145150 155 GCA GCA GTG ACC GGA TCA TCT CTC TCA ACT TCC GAG GCA CAG GAT GCA586 Ala Ala Val Thr Gly Ser Ser Leu Ser Thr Ser Glu Ala Gln Asp Ala 160165 170 175 GGG CTT ACG GCT AAG CCT CAG AGC ATT GGA AGT TTT GAG GCG GCTGAC 634 Gly Leu Thr Ala Lys Pro Gln Ser Ile Gly Ser Phe Glu Ala Ala Asp180 185 190 ATC TCC ACC ACC GTT TGG CCG AGT CCT GCT GTC TAC CAA TCT GGATCT 682 Ile Ser Thr Thr Val Trp Pro Ser Pro Ala Val Tyr Gln Ser Gly Ser195 200 205 AGC TCC TGG GCT GAG GAA AAA GCT ACT GAG TCC CCC TCC ACT ACAGCC 730 Ser Ser Trp Ala Glu Glu Lys Ala Thr Glu Ser Pro Ser Thr Thr Ala210 215 220 CCA TCT CCT CAG GTG TCC ACT ACT TCA CCT TCA ACC CCA GAG GAAAAT 778 Pro Ser Pro Gln Val Ser Thr Thr Ser Pro Ser Thr Pro Glu Glu Asn225 230 235 GTT GGG TCC GAA GGC CAA CCC CCA TGG GTC CAG GGA CAG GAC CTCAGT 826 Val Gly Ser Glu Gly Gln Pro Pro Trp Val Gln Gly Gln Asp Leu Ser240 245 250 255 CCA GAG AAG TCT CTA GGG TCT GAG GAG ATA AAC CCA GTT CATACT GAT 874 Pro Glu Lys Ser Leu Gly Ser Glu Glu Ile Asn Pro Val His ThrAsp 260 265 270 AAT TTC CAG GAG AGG GGG CCT GGC AAC ACA GTC CAC CCC TCAGTG GCT 922 Asn Phe Gln Glu Arg Gly Pro Gly Asn Thr Val His Pro Ser ValAla 275 280 285 CCC ATC TCC TCT GAA GAG ACC CCC AGC CCA GAG CTG GTG GCCTCG GGC 970 Pro Ile Ser Ser Glu Glu Thr Pro Ser Pro Glu Leu Val Ala SerGly 290 295 300 AGC CAG GCT CCT AAG ATA GAG GAA CCC ATC CAT GCC ACT GCAGAT CCC 1018 Ser Gln Ala Pro Lys Ile Glu Glu Pro Ile His Ala Thr Ala AspPro 305 310 315 CAG AAA CTG AGT GTG CTT ATC ACT CCT GTC CCC GAC ACC CAGGCA GCC 1066 Gln Lys Leu Ser Val Leu Ile Thr Pro Val Pro Asp Thr Gln AlaAla 320 325 330 335 ACA AGG AGG CAG GCA GTG GGG CTA CTG GCT TTC CTT GGTCTT CTT TTC 1114 Thr Arg Arg Gln Ala Val Gly Leu Leu Ala Phe Leu Gly LeuLeu Phe 340 345 350 TGC CTA GGG GTG GCC ATG TTT GCT TAC CAG AGC CTT CAGGGC TGT CCC 1162 Cys Leu Gly Val Ala Met Phe Ala Tyr Gln Ser Leu Gln GlyCys Pro 355 360 365 CGC AAA ATG GCG GGG GAA ATG GTA GAA GGC CTC CGC TACGTC CCC CGT 1210 Arg Lys Met Ala Gly Glu Met Val Glu Gly Leu Arg Tyr ValPro Arg 370 375 380 AGC TGT GGC AGT AAC TCA TAC GTC CTG GTG CCA GTG TGAGCTGCTTGCC 1259 Ser Cys Gly Ser Asn Ser Tyr Val Leu Val Pro Val * 385390 395 TGCCTGCCTG TGTCCAGAGT GTGATTCGGA CAGCTGTCTG GGGACCCCCCCCCATCCTCA 1319 TACCCACCTT CATCCACGCT GGGGAAATGG GAATGGAGAA GCTGGACCTCCAGGGGCTGT 1379 GGGCTCCATC CAATCCCCCT TCCCCCGAGG GGTGGCCCCG GAGGCCACCCTAGACCACTA 1439 TTCACTTATC AGAGACAGAG CAGGTGACCT TCCAGCTCCT CTATATTTGAAAGAATCCTC 1499 TGCTGCTGGC TGGTTAGAGG GGCCCTTGAC ACCCCAACTC CAGTGAACAATTATTTATTG 1559 GATTCCCAGC CCCTGCGACG ACACCTGTTT CCCGCGCGCA CCGTGGTCCGCCCATATCAC 1619 AAGCAGCAGG CCAGGCCTAT CTGCCTGTCC CCCTGACCTC CTTGTGTCTCCTGGCTTTGC 1679 TGCAGTCGCC AGCCCTTCTC CTCCCCGGCC AGCCGCGGTG CTATCTGCCCTATGTCTCCC 1739 TCTATCCCCT GTACAGAGCG CACCACCATC ACCATCAACA CCGCTGTTGTGTCTTTTCTT 1799 GCATGAGGTT AAAGCTGTGT TTTCTGGAGC TCTCCGGGAA GGGAGACAAGCTTGCGAGAG 1859 GGTTTAAAGT GTTCCTCCCC AGACTTGGAT GTGCTGTGAG GGCATGCTGCGTCTGAAGGA 1919 AGGGTCCAGT CCCCACTCGG CTACCAGCAC CACAAAGTGC CCCACCTGTAAAAGGAAAGA 1979 AACGTGGTCC AGAGCTGGCA ATAACCTATG GCCCTGACAT CATCACTTTCTCTGAGATCC 2039 TTGTCTCCAC CCCTGGGTGC AACCCCACCC CTTATCAACA TTAATAGTCACTGCCATTCC 2099 ACTGGACTGA CATTTTTGTA CCCTGTGATT CTGAGGGCTG GCAAGGAGTGGCTTGAGAGT 2159 GCAGATCGTA CCCTGTATGC CCCCCCCAAA TGGAGGCTGA GTTGGGGACTTGCAGGAACA 2219 GAGGCCAACT CAGATGGCTT CCCCTGTGTT CTCACTAGAA ACCCCTCCCCCATGCACCAA 2279 GGTGACAGTC ACAGGTCTGC CCTGGCTAAA GGACAAGCCA CATAGGAAAGATTAGGACAA 2339 GCCCCTCGGA GGCAGAGGAT CCAGGGTAAA CCCCTGGAGT GGCCACAAACCCAATTTCAG 2399 TGTAGGGACT TGTGCATGTG TGTACTTGCA TAGTCAGACA GAGGCTGCCAGGGTCCTTTC 2459 CTGTCTCTGA GAGCAGTGTT CACGCCAAGG ACTCACCTTT GCCCCCATTGCAGGCAGGGC 2519 CAGAACTCCC ATAGCATTCT CCAAGAGCCC TGTGACATTT TCTGGAAGGAACTCTGCCCT 2579 GGGCGCAAAG TGACTGCTGA AGCAAGGAGC AGCTGAGCAG CACCCCAGCGGAGCTGAGCC 2639 GGCAGGCCAC GCCCCTCGGG GGGGGGCATT TCTACCCGCC CTGCTCTGAATAGCTCCAAC 2699 TTCACCTTAG GAGCCTCCCA GGGGCGAGCT TCACCCAGAA GCCAGTGACTCACTCCTTGA 2759 TTGGTGGAAG CTCAGTTGGC TCCTGAGAGT GAGGAAGCCA ACCCTTTGTCGACCCTCCTC 2819 CTGGGAAGCC TGTGGGCGGC TCTGATCATG CTCCACAGAA CCAGTTGTAGGCCTGAGCCG 2879 CAGCAGCCCG AGTGCACTAT ATCCTGGCTC CTTCGGTGGG GAACCTTTAAGGGTTGGGAC 2939 ACCCGTCATC GGACTTTGTT GGTTCCTCCC TCCCAGAGCA GAATGTGGGCCGTAACAATC 2999 TGAGGAGGAC TTTAAAAGTT GTTGATCCTT TAGGGTTTTT TTTCAAGCATCATTACCAAT 3059 GTCTGT 3065 395 amino acids amino acid linear protein 8Met Ala Pro Ser Pro Leu Ala Trp Leu Leu Arg Leu Ala Ala Phe Phe 1 5 1015 His Leu Cys Thr Leu Leu Pro Gly Gln His Leu Gly Met Thr Lys Cys 20 2530 Glu Ile Met Cys Gly Lys Met Thr Ser Arg Ile Pro Val Ala Leu Leu 35 4045 Ile Arg Tyr Gln Leu Asn Gln Glu Ser Cys Gly Lys Arg Ala Ile Val 50 5560 Leu Glu Thr Thr Gln His Arg Arg Phe Cys Ala Asp Pro Lys Glu Lys 65 7075 80 Trp Val Gln Asp Ala Met Lys His Leu Asp His Gln Ala Ala Ala Leu 8590 95 Thr Lys Asn Gly Gly Lys Phe Glu Lys Arg Val Asp Asn Val Thr Pro100 105 110 Gly Ile Thr Leu Ala Thr Arg Gly Leu Ser Pro Ser Ala Leu ThrLys 115 120 125 Pro Glu Ser Ala Thr Leu Glu Asp Leu Ala Leu Glu Leu ThrThr Ile 130 135 140 Ser Gln Glu Ala Arg Gly Thr Met Gly Thr Ser Gln GluPro Pro Ala 145 150 155 160 Ala Val Thr Gly Ser Ser Leu Ser Thr Ser GluAla Gln Asp Ala Gly 165 170 175 Leu Thr Ala Lys Pro Gln Ser Ile Gly SerPhe Glu Ala Ala Asp Ile 180 185 190 Ser Thr Thr Val Trp Pro Ser Pro AlaVal Tyr Gln Ser Gly Ser Ser 195 200 205 Ser Trp Ala Glu Glu Lys Ala ThrGlu Ser Pro Ser Thr Thr Ala Pro 210 215 220 Ser Pro Gln Val Ser Thr ThrSer Pro Ser Thr Pro Glu Glu Asn Val 225 230 235 240 Gly Ser Glu Gly GlnPro Pro Trp Val Gln Gly Gln Asp Leu Ser Pro 245 250 255 Glu Lys Ser LeuGly Ser Glu Glu Ile Asn Pro Val His Thr Asp Asn 260 265 270 Phe Gln GluArg Gly Pro Gly Asn Thr Val His Pro Ser Val Ala Pro 275 280 285 Ile SerSer Glu Glu Thr Pro Ser Pro Glu Leu Val Ala Ser Gly Ser 290 295 300 GlnAla Pro Lys Ile Glu Glu Pro Ile His Ala Thr Ala Asp Pro Gln 305 310 315320 Lys Leu Ser Val Leu Ile Thr Pro Val Pro Asp Thr Gln Ala Ala Thr 325330 335 Arg Arg Gln Ala Val Gly Leu Leu Ala Phe Leu Gly Leu Leu Phe Cys340 345 350 Leu Gly Val Ala Met Phe Ala Tyr Gln Ser Leu Gln Gly Cys ProArg 355 360 365 Lys Met Ala Gly Glu Met Val Glu Gly Leu Arg Tyr Val ProArg Ser 370 375 380 Cys Gly Ser Asn Ser Tyr Val Leu Val Pro Val 385 390395 96 amino acids amino acid single linear peptide 9 Met Ile Pro AlaThr Arg Ser Leu Leu Cys Ala Ala Leu Leu Leu Leu 1 5 10 15 Ala Thr SerArg Leu Ala Thr Gly Ala Pro Ile Ala Asn Glu Leu Arg 20 25 30 Cys Gln CysLeu Gln Thr Met Ala Gly Ile His Leu Lys Asn Ile Gln 35 40 45 Ser Leu LysVal Leu Pro Ser Gly Pro His Cys Thr Gln Thr Glu Val 50 55 60 Ile Ala ThrLeu Lys Asn Gly Arg Glu Ala Cys Leu Asp Pro Glu Ala 65 70 75 80 Pro LeuVal Gln Lys Ile Val Gln Lys Met Leu Lys Gly Val Pro Lys 85 90 95 115amino acids amino acid single linear peptide 10 Met Arg Leu Leu Leu LeuThr Phe Leu Gly Val Cys Cys Leu Thr Pro 1 5 10 15 Trp Val Val Glu GlyVal Gly Thr Glu Val Leu Glu Glu Ser Ser Cys 20 25 30 Val Asn Leu Gln ThrGln Arg Leu Pro Val Gln Lys Ile Lys Thr Tyr 35 40 45 Ile Ile Trp Glu GlyAla Met Arg Ala Val Ile Phe Val Thr Lys Arg 50 55 60 Gly Leu Lys Ile CysAla Asp Pro Glu Ala Lys Trp Val Leu Ala Ala 65 70 75 80 Ile Lys Thr ValAsp Gly Arg Ala Ser Thr Arg Lys Asn Met Ala Glu 85 90 95 Thr Val Pro GlyThr Gly Ala Gln Arg Ser Thr Ser Thr Ala Ile Thr 100 105 110 Leu Thr Gly115 92 amino acids amino acid single linear peptide 11 Met Lys Leu CysVal Ser Ala Leu Ser Leu Leu Leu Leu Val Ala Ala 1 5 10 15 Phe Cys AlaPro Gly Phe Ser Ala Pro Met Gly Ser Asp Pro Pro Thr 20 25 30 Ser Cys CysPhe Ser Tyr Thr Ala Arg Lys Leu Pro Arg Asn Phe Val 35 40 45 Val Asp TyrTyr Glu Thr Ser Ser Leu Cys Ser Gln Pro Ala Val Val 50 55 60 Phe Gln ThrLys Arg Ser Lys Gln Val Cys Ala Asp Pro Ser Glu Ser 65 70 75 80 Trp ValGln Glu Tyr Val Tyr Asp Leu Glu Leu Asn 85 90

What is claimed is:
 1. A composition of matter selected from the groupcomprising: a) an antibody binding site which specifically binds to amammalian CX3C chemokine; b) an expression vector encoding a mammalianCX3C chemokine or fragment thereof; c) a substantially pure proteinwhich is specifically recognized by said antibody binding site of (a);and d) a substantially pure CX3C chemokine or polypeptide thereof, or afusion protein comprising a 30 amino acid sequence portion of CX3Cchemokine sequence.
 2. The composition of claim 1, wherein said antibodybinding site is: a) specifically immunoreactive with a protein selectedfrom the group consisting of the polypeptides of SEQ ID NO 2, 4, 6 and8; b) raised against a purified or recombinantly produced human or mouseCX3C chemokine; c) in a monoclonal antibody, Fab, or F(ab)2; d) able tobind a denatured antigen; or e) in a labeled antibody.
 3. The antibodybinding site of claim 1, wherein said binding site is detected in abiological sample by a method comprising the steps of: a) contacting abinding agent having an affinity for said CX3C chemokine protein withsaid biological sample; b) incubating said binding agent with saidbiological sample to form a binding agent:CX3C chemokine proteincomplex; and c) detecting said complex.
 4. The method of claim 3,wherein said biological sample is human, and wherein said binding agentis an antibody.
 5. A kit comprising a composition of claim 1, whereinsaid kit further comprises: a) instructional material for the use ofsaid composition; or b) segregation of said composition into acontainer.
 6. The vector of claim 1, encoding a CX3C chemokine protein,wherein said protein specifically binds an antibody generated against animmunogen selected from the group consisting of the mature polypeptidesof SEQ ID NO 2, 4, 6 and
 8. 7. The vector of claim 1, wherein saidvector: a) encodes a CX3C chemokine polypeptide with complete sequenceidentity to a naturally occurring chemokine domain of a human CX3Cchemokine protein; b) encodes a CX3C chemokine protein comprisingsequence selected from the group consisting of the mature polypeptidesof SEQ ID NO 2, 4, 6, and 8; or c) comprises sequence selected from thegroup consisting of the nucleic acids of SEQ ID NO 1, 3, 5, and
 7. 8.The vector of claim 1, wherein said vector is capable of selectivelyhybridizing to a nucleic acid encoding a CX3C chemokine protein.
 9. Thevector of claim 1, which comprises a mature protein coding segment ofSEQ ID NO 1, 3, 5, or
 7. 10. The vector of claim 1, wherein saidisolated nucleic acid is detected in a biological sample by a methodcomprising: a) contacting said biological sample with a nucleic acidprobe capable of selectively hybridizing to said nucleic acid; b)incubating said nucleic acid probe with the biological sample to form ahybrid of the nucleic acid probe with complementary nucleic acidsequences present in the biological sample; and c) determining theextent of hybridization of the nucleic acid probe to the complementarynucleic acid sequences.
 11. The method of claim 10, wherein said nucleicacid probe is capable of hybridizing to a nucleic acid encoding aprotein selected from the group consisting of the mature polypeptides ofSEQ ID NO 2, 4, 6, and
 8. 12. An isolated CX3C chemokine protein of lessthan 15,000 daltons when in unglycosylated form, wherein said CX3Cchemokine protein specifically binds to an antibody generated against animmunogen selected from the group consisting of: a) the polypeptide ofSEQ ID NO: 2; b) the polypeptide of SEQ ID NO: 4; c) the polypeptide ofSEQ ID NO: 6; and d) the polypeptide of SEQ ID NO: 8 and said CX3Cchemokine lacks the cysteine structural motifs and sequencecharacteristic of a C, a CC, or a CXC chemokine.
 13. The protein ofclaim 1, wherein said isolated CX3C chemokine polypeptide: a) isselected from the group consisting of human CX3Ckine and mouse CX3Ckine;b) comprises mature polypeptide sequence selected from the groupconsisting of SEQ ID NO 2, 4, 6, and 8; c) consists essentially of thechemokine domain; or d) is a soluble protein.
 14. The isolated CX3Cchemokine protein of claim 1, wherein said isolated CX3C chemokineprotein is: a) recombinantly produced, or b) a naturally occurringprotein.
 15. The fusion protein of claim 1, which comprises sequencefrom the chemokine, stalk, or intracellular domains of human or mouseCX3C chemokine of SEQ ID NO: 4 or
 8. 16. A cell transfected with thenucleic acid of claim
 1. 17. The cell of claim 16, wherein said nucleicacid consists of a polynucleotide sequence selected from the groupconsisting of the nucleic acids of SEQ ID NO 1, 3, 5 and
 7. 18. A methodof modulating physiology or development of a cell comprising contactingsaid cell with a CX3C chemokine, or an antagonist of said chemokine. 19.The method of claim 17, wherein said physiology is attraction, and saidcell is a peripheral blood monocyte or a T cell.
 20. A sterilecomposition of matter of claim
 1. 21. A method of making a CX3Ckinepolypeptide comprising expressing a vector of claim 1.